WO2025110703A1 - Pharmaceutical composition for preventing or treating diseases caused by grp78 overexpression, comprising homoharringtonine as active ingredient - Google Patents
Pharmaceutical composition for preventing or treating diseases caused by grp78 overexpression, comprising homoharringtonine as active ingredient Download PDFInfo
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- WO2025110703A1 WO2025110703A1 PCT/KR2024/018369 KR2024018369W WO2025110703A1 WO 2025110703 A1 WO2025110703 A1 WO 2025110703A1 KR 2024018369 W KR2024018369 W KR 2024018369W WO 2025110703 A1 WO2025110703 A1 WO 2025110703A1
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the present invention relates to a pharmaceutical composition for preventing or treating a disease caused by GRP78 (Glucose-Regulated Protein 78) overexpression, which contains homoharringtonine as an active ingredient.
- GRP78 Glucose-Regulated Protein 78
- GRP78 Glucose-Regulated Protein 78
- GRP78 is a protein consisting of 654 amino acids, and is composed of three domains: an N-terminal nucleotide domain with ATPase activity, a substrate binding domain, and a variable C-terminal domain.
- the substrate binding specificity and activity of GRP78 are related to the ATPase activity of GRP78.
- HA15 is a commercially available GRP78 inhibitor that is known to bind to GRP78 and inhibit the ATPase activity of GRP78.
- homoharringtonine Cephalotaxus It is a natural single component isolated from hainanensis and is commercially used as an adjuvant treatment for chronic myeloid leukemia. Its mechanism of action is known to inhibit protein production by binding to 80S ribosomes in eukaryotic cells and inhibiting chain elongation.
- homoharringtonine and GRP78 has not been reported yet.
- the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating a disease caused by GRP78 (Glucose-Regulated Protein 78) overexpression.
- GRP78 Glucose-Regulated Protein 78
- Another object of the present invention is to provide a health functional food composition for preventing or improving a disease caused by overexpression of GRP78 (Glucose-Regulated Protein 78).
- GRP78 Glucose-Regulated Protein 78
- Another object of the present invention is to provide a method for preventing or treating a disease caused by GRP78 overexpression, comprising a step (first step) of treating homoharringtonine in a patient group having a high expression of GRP78 (Glucose-Regulated Protein 78) compared to a general patient group in a population other than humans.
- GRP78 Glucose-Regulated Protein 78
- the present invention provides a pharmaceutical composition for preventing or treating a disease caused by GRP78 (Glucose-Regulated Protein 78) overexpression, which contains homoharringtonine as an active ingredient.
- GRP78 Glucose-Regulated Protein 78
- the present invention provides a health functional food composition for preventing or improving a disease caused by GRP78 (Glucose-Regulated Protein 78) overexpression, which contains homoharringtonine as an active ingredient.
- GRP78 Glucose-Regulated Protein 78
- the present invention provides a method for preventing or treating a disease caused by GRP78 overexpression, including a step (first step) of treating homoharringtonine in a patient group having a high expression of GRP78 (Glucose-Regulated Protein 78) compared to a general patient group in a population other than humans.
- GRP78 Glucose-Regulated Protein 78
- the present invention provides a reagent composition for inhibiting GRP78 (Glucose-Regulated Protein 78) expression, which contains homoharringtonine as an active ingredient.
- GRP78 Glucose-Regulated Protein 78
- Figure 1 shows the results of analyzing the effect of homoharringtonine (hereinafter referred to as HHT) on ATPase activity. *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
- Figure 2 shows the results of analyzing the effect of HHT on GRP78 (Glucose-Regulated Protein 78) expression. *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
- the present invention provides a pharmaceutical composition for preventing or treating a disease caused by GRP78 (Glucose-Regulated Protein 78) overexpression, which contains homoharringtonine as an active ingredient.
- GRP78 Glucose-Regulated Protein 78
- the above homoharringtonine can inhibit ATPase activity.
- GRP78 binds and maintains the transmembrane ER stress sensor in an inactive form, and GRP78 is released when ER stress occurs.
- tunicamycin an ER stress inducer, induces ER stress in cells by generating a large amount of misfolded proteins, thereby increasing the protein expression of GRP78.
- the above disease may be one or more selected from the group consisting of, but is not limited to, multiple myeloma, lung cancer, pancreatic cancer, colon cancer, breast cancer, nasopharyngeal cancer, kidney cancer, glioblastoma, ovarian cancer, liver cancer, biliary tract cancer, osteosarcoma, papillary thyroid cancer, stomach cancer, esophageal cancer, prostate cancer, tongue cancer, and Wilms tumor.
- the pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains.
- the pharmaceutically acceptable carriers mentioned above are those commonly used in the preparation of formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.
- the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.
- the content of the additive included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.
- the above pharmaceutical composition may be formulated in the form of one or more skin external preparations selected from the group consisting of injectable preparations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, pastes, lotions, liniments, pastes, and cataplasmas, but is not limited thereto.
- injectable preparations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, pastes, lotions, liniments, pastes, and cataplasmas, but is not limited thereto.
- the pharmaceutical composition of the present invention may further comprise pharmaceutically acceptable carriers and diluents for formulation.
- the pharmaceutically acceptable carriers and diluents include, but are not limited to, excipients such as starch, sugar and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose, binders such as gelatin, alginates and polyvinyl pyrrolidone, lubricants such as talc, calcium stearate, hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone and crospovidone, and surfactants such as polysorbates, cetyl alcohol, glycerol and the like.
- the pharmaceutically acceptable carriers and diluents may be biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents and/or dispersion media.
- the pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method.
- oral administration it may be formulated as tablets, troches, lozenges, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, elixirs, etc.
- parenteral administration it may be formulated as injections, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for application, oils, creams, etc.
- the dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition, weight, age, sex, health condition, dietary constitution, nature of the preparation, degree of disease, administration time of the composition, administration method, administration period or interval, excretion rate, and drug form, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg/kg, but is not limited thereto, and may be administered once a day or divided into several times.
- the present invention provides a health functional food composition for preventing or improving a disease caused by GRP78 (Glucose-Regulated Protein 78) overexpression, which contains homoharringtonine as an active ingredient.
- GRP78 Glucose-Regulated Protein 78
- the present invention can be generally used as a commonly used food.
- the food composition of the present invention can be used as a health functional food.
- the above “health functional food” means a food manufactured and processed using raw materials or ingredients having functionality useful to the human body according to the Health Functional Food Act, and “functionality” means that it is consumed for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects.
- the above health functional food composition may contain conventional food additives, and its suitability as the above “food additive” is determined by the specifications and standards for the relevant item according to the general provisions and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.
- Food Additives Codex include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular-weight pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents added to noodles, preservative preparations, and tar color preparations.
- chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid
- natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular-weight pigment, and guar gum
- mixed preparations such as sodium L-glutamate preparations, alkaline agents added to noodles, preservative preparations, and tar color preparations.
- the food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc.
- hard capsules can be manufactured by mixing and filling a composition according to the present invention with additives such as excipients into a conventional hard capsule
- soft capsules can be manufactured by mixing a composition according to the present invention with additives such as excipients and filling a capsule base such as gelatin.
- the soft capsules can contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., as necessary.
- prevention in the present invention refers to any act of suppressing or delaying a disease caused by GRP78 overexpression by administering a composition according to the present invention.
- treatment refers to any act of improving or beneficially changing the symptoms of a disease caused by GRP78 overexpression by administering a composition according to the present invention.
- improvement in the present invention refers to any act of improving the bad condition of a disease caused by GRP78 overexpression by administering a composition according to the present invention.
- the present invention provides a method for preventing or treating a disease caused by GRP78 overexpression, including a step (first step) of treating homoharringtonine in a patient group having a high expression of GRP78 (Glucose-Regulated Protein 78) compared to a general patient group in a population other than humans.
- GRP78 Glucose-Regulated Protein 78
- the present invention provides a reagent composition for inhibiting GRP78 (Glucose-Regulated Protein 78) expression, which contains homoharringtonine as an active ingredient.
- GRP78 Glucose-Regulated Protein 78
- HDF cells Human dermal fibroblast cells
- ATCC Manassas, VA, USA
- the cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) (WelGENE, Gyeongsan-si, Korea) supplemented with 10% FBS (fetal bovine serum) (GIBCO, NE, USA) and 1 ⁇ penicillin-streptomycin (Hyclone, Logan, UT, USA) at 37°C and 5% CO 2.
- FBS fetal bovine serum
- Hyclone fetal bovine serum
- the cells were then treated with HHT (100 nM, TOCRIS, UK) and cultured for 2 hours, then treated with tunicamycin (2 ⁇ g/mL), and cultured overnight.
- HHT human dermal fibroblast cells
- Triple-negative breast cancer cells (MDA-MB-231 and HCC-1937) were purchased from ATCC (Manassas, VA, USA). Cells were cultured in RPMI1640 (Roswell Park Memorial Institute 1640) (WelGENE, Gyeongsan-si, Korea) supplemented with 10% FBS (GIBCO, NE, USA) and 1 ⁇ penicillin-streptomycin (GIBCO, NE, USA) at 37°C and 5% CO 2 .
- RPMI1640 Roswell Park Memorial Institute 1640
- FBS GIBCO, NE, USA
- penicillin-streptomycin NE, USA
- mice Eight-week-old C57BL/6 male mice (KOATECH, Pyeongtaek-si, Korea) were acclimated to the breeding environment for 1 week.
- the experimental groups were set up as follows. The mice were divided into a normal diet group and a high-fat diet group, and the high-fat diet group was fed a diet in which 60.3% of the total calories were fat (#TD.06414, R&D systems, Minneapolis, MN, USA) for 6 weeks. After that, the high-fat diet group was divided into a control group and an experimental group, and the control group was administered PBS and the experimental group was administered HHT intraperitoneally.
- HHT human immunodeficiency virus
- DMSO Dimethyl sulfoxide
- PBS PBS
- the HHT diluted in PBS was administered 4 ⁇ l (0.545 ⁇ g of HHT) per g of mouse body weight. Therefore, 0.545 ⁇ g/g body weight/time per mouse was administered intraperitoneally three times a week for 8 weeks (total administration: 13.08 ⁇ g/g body weight).
- the normal group was also administered intraperitoneally PBS containing the same amount of DMSO (total administration: 96 ⁇ l/g body weight).
- mice were raised in an environment with a controlled room temperature of 22 ⁇ 2°C and a 12-h light/dark cycle until the end of the experiment, and their body weight and food intake were measured at weekly intervals. After the end of the experiment, the mice were anesthetized with Avertin anesthesia, and their adipose tissue was collected and stored at -80°C until analysis.
- Control group High-fat diet + PBS (96 ⁇ l/g body weight) administration
- mice 18-month-old C57BL/6 mice (KOATECH, Pyeongtaek-si, Korea) were acclimated to the breeding environment for 1 week.
- HHT was dissolved in DMSO in the same manner as in Example 1-2, and diluted with PBS for use.
- the experimental groups were set up as follows. The mice were divided into a control group and an experimental group. The control group was administered PBS and the experimental group was administered HHT (0.545 ⁇ g/g body weight/once) intraperitoneally three times a week for a total of 19 weeks (total dosage: 31.065 ⁇ g/g body weight).
- the control group was administered PBS (4 ⁇ l/g body weight/once) intraperitoneally three times a week for a total of 19 weeks (total dosage: 228 ⁇ g/g body weight).
- the mice were raised in an environment controlled at an indoor temperature of 22 ⁇ 2°C and a 12-hour light/dark cycle until the end of the experiment, and their body weight and food intake were measured at weekly intervals. After the experiment, the mice were anesthetized with Avertin, and the tibialis anterior muscle was collected and stored at -80°C until analysis.
- Control group PBS (228 ⁇ l/g body weight) administered
- HHT HHT (31.065 ⁇ g/g body weight) administered
- ATPase activity was measured using an ATPase Activity Assay Kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) according to the manufacturer's instructions.
- 40 mM Tris, 80 mM NaCl, 8 mM MgAc 2 , 1 mM EDTA, and 4 mM ATP were mixed (pH 7.5), and GRP78 protein (5 ⁇ g); GRP78 protein (5 ⁇ g) + HHT (10 ⁇ M); or GRP78 protein (5 ⁇ g) + HA15 (10 ⁇ M, HTT inhibitor, positive control) were mixed to prepare a reaction mixture, and then incubated at room temperature for 60 min.
- reagent [reagent (MAK113A)] included in the kit was added to each well, and the enzyme reaction was completed by incubating for an additional 10 to 30 minutes at room temperature, and a colorimetric product was generated.
- the colorimetric product was transferred to a 96-well plate, and the phosphate activity (absorbance) at 620 nm was measured using a microplate reader (SunriseTM, TECAN, Switzerland). A standard curve was established using phosphate standards.
- a lysis buffer containing protease inhibitor [150 mM NaCl, 50 mM HEPES, 50 mM sodium fluoride (NaF), 1 mM benzamide, 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1% NP-40, 10% glycerol, 0.22% ⁇ -glycerophosphate] was added to a certain amount of the animal tissues collected and rapidly frozen in the above Experimental Examples 1-2 and 1-3, homogenized using microbeads, and centrifuged at 13,000 rpm at 4°C. Protein was extracted.
- protease inhibitor 150 mM NaCl, 50 mM HEPES, 50 mM sodium fluoride (NaF), 1 mM benzamide, 1 mM EGTA, 1 mM EDTA, 1 mM
- the above proteins were quantified, and after performing SDS-PAGE, the proteins were transferred to a membrane. After that, primary and secondary antibodies were reacted in sequence, and chemiluminescence was confirmed using an ECL kit (Amersham Biosciences, NJ, USA). The expressed bands were quantified using the ImageJ (National Institute of Mental Health, Bethesda, Maryland, USA) program. The protein levels of each experimental group were expressed by correcting them to the GAPDH protein level, which is a housekeeping protein of each experimental group.
- ATPase activity was significantly reduced in the HHT treatment group (GRP78+HHT) and the positive control group (GRP78+HA15) compared to the GRP78 only treatment group (GRP78). Specifically, HHT reduced ATPase activity by approximately 30%, and the positive control group, HA15, reduced ATPase activity by approximately 20%. From the above results, it was confirmed that HHT inhibits ATPase activity.
- GRP78 expression was significantly higher in MDA-MB-231 cells than in HCC-1937 cells. From the results above, it was confirmed that there was a difference in GRP78 expression depending on the breast cancer cell type.
- HHT human epithelial growth factor
- TCT triple-negative breast cancer cells cultured in Experimental Example 1-1 were treated with HHT (100 nM, TOCRIS, UK) and cultured for 3 days. Then, 10 ⁇ L of WST reagent (EZ-Cytox, DoGenBio, seoul, Korea) was treated to 200 ⁇ L of the cultured medium, and after reacting for 2 to 3 hours and 30 minutes, the cell viability was confirmed by measuring the absorbance at 450 nm using a microplate reader (SunriseTM, TECAN, Switzerland).
- the anticancer effect (cell growth inhibition effect) of HHT was more significant in MDA-MB-231 cells with high GRP78 expression than in HCC-1937 cells with low GRP78 expression.
- the cell viability rates of MDA-MB-231 cells and HCC-1937 cells in the HHT treatment group were approximately 20.5% and 47.4%, respectively. From the above results, it was confirmed that HHT had a more excellent anticancer effect on breast cancer with high GRP78 expression than on breast cancer with low GRP78 expression.
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Abstract
Description
본 발명은 호모해링토닌(homoharringtonine)을 유효성분으로 포함하는 GRP78(Glucose-Regulated Protein 78) 과발현에 의한 질환 예방 또는 치료용 약학 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating a disease caused by GRP78 (Glucose-Regulated Protein 78) overexpression, which contains homoharringtonine as an active ingredient.
GRP78(Glucose-Regulated Protein 78)은 654개의 아미노산으로 이루어진 단백질로, ATPase 활성이 있는 N-terminal의 뉴클레오타이드 도메인, 기질 결합 도메인 및 variable C-terminal 도메인으로, 총 3개의 도메인으로 구성되어 있다. GRP78의 기질 결합 특이성과 활성은 GRP78의 ATPase 활성과 연관되어 있다. HA15는 현재 시판되고 있는 GRP78 억제제로 GRP78과 결합하여 GRP78의 ATPase 활성을 억제한다고 알려져 있다.GRP78 (Glucose-Regulated Protein 78) is a protein consisting of 654 amino acids, and is composed of three domains: an N-terminal nucleotide domain with ATPase activity, a substrate binding domain, and a variable C-terminal domain. The substrate binding specificity and activity of GRP78 are related to the ATPase activity of GRP78. HA15 is a commercially available GRP78 inhibitor that is known to bind to GRP78 and inhibit the ATPase activity of GRP78.
한편, 호모해링토닌은 Cephalotaxus hainanensis에서 분리된 천연물 단일성분으로, 만성골수성백혈병(chronic myeloid leukemia) 보조 치료제로 상용되고 있다. 그 작용 기전은 진핵세포에서 80S 리보솜과 결합하여 사슬 연장을 저해함으로써 단백질 생성을 억제한다고 알려져 있다. 그러나 아직까지 호모해링토닌 및 GRP78의 연관성에 대해서는 보고된 바 없다.Meanwhile, homoharringtonine Cephalotaxus It is a natural single component isolated from hainanensis and is commercially used as an adjuvant treatment for chronic myeloid leukemia. Its mechanism of action is known to inhibit protein production by binding to 80S ribosomes in eukaryotic cells and inhibiting chain elongation. However, the relationship between homoharringtonine and GRP78 has not been reported yet.
본 발명의 목적은 GRP78(Glucose-Regulated Protein 78) 과발현에 의한 질환 예방 또는 치료용 약학 조성물을 제공하는 것이다.The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating a disease caused by GRP78 (Glucose-Regulated Protein 78) overexpression.
본 발명의 다른 목적은 GRP78(Glucose-Regulated Protein 78) 과발현에 의한 질환 예방 또는 개선용 건강기능식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a health functional food composition for preventing or improving a disease caused by overexpression of GRP78 (Glucose-Regulated Protein 78).
본 발명의 또 다른 목적은 인간을 제외한 개체군에 있어서, 일반 환자군 대비 GRP78(Glucose-Regulated Protein 78) 발현이 높은 환자군에 호모해링토닌(homoharringtonine)을 처리하는 단계(제1단계)를 포함하는, GRP78 과발현에 의한 질환 예방 또는 치료 방법을 제공하는 것이다.Another object of the present invention is to provide a method for preventing or treating a disease caused by GRP78 overexpression, comprising a step (first step) of treating homoharringtonine in a patient group having a high expression of GRP78 (Glucose-Regulated Protein 78) compared to a general patient group in a population other than humans.
본 발명의 또 다른 목적은 GRP78(Glucose-Regulated Protein 78) 발현 억제용 시약 조성물을 제공하는 것이다.Another object of the present invention is to provide a reagent composition for inhibiting GRP78 (Glucose-Regulated Protein 78) expression.
상기 목적을 달성하기 위해, 본 발명은 호모해링토닌(homoharringtonine)을 유효성분으로 포함하는 GRP78(Glucose-Regulated Protein 78) 과발현에 의한 질환 예방 또는 치료용 약학 조성물을 제공한다.To achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating a disease caused by GRP78 (Glucose-Regulated Protein 78) overexpression, which contains homoharringtonine as an active ingredient.
또한, 본 발명은 호모해링토닌(homoharringtonine)을 유효성분으로 포함하는 GRP78(Glucose-Regulated Protein 78) 과발현에 의한 질환 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or improving a disease caused by GRP78 (Glucose-Regulated Protein 78) overexpression, which contains homoharringtonine as an active ingredient.
또한, 본 발명은 인간을 제외한 개체군에 있어서, 일반 환자군 대비 GRP78(Glucose-Regulated Protein 78) 발현이 높은 환자군에 호모해링토닌(homoharringtonine)을 처리하는 단계(제1단계)를 포함하는, GRP78 과발현에 의한 질환 예방 또는 치료 방법을 제공한다.In addition, the present invention provides a method for preventing or treating a disease caused by GRP78 overexpression, including a step (first step) of treating homoharringtonine in a patient group having a high expression of GRP78 (Glucose-Regulated Protein 78) compared to a general patient group in a population other than humans.
또한, 본 발명은 호모해링토닌(homoharringtonine)을 유효성분으로 포함하는 GRP78(Glucose-Regulated Protein 78) 발현 억제용 시약 조성물을 제공한다.In addition, the present invention provides a reagent composition for inhibiting GRP78 (Glucose-Regulated Protein 78) expression, which contains homoharringtonine as an active ingredient.
본 발명에 따르면, 호모해링토닌(homoharringtonine)이 GRP78(Glucose-Regulated Protein 78) 발현을 억제하고, GRP78 발현이 낮은 유방암 대비 GRP78 발현이 높은 유방암에서 호모해링토닌의 항암 효과가 더 유의하게 나타나는 것을 확인함으로써, GRP78 과발현에 의한 질환 예방, 치료 또는 개선용 조성물; 상기 질환 예방 또는 치료 방법; 또는 GRP78 발현 억제용 시약 조성물로써 유용하게 활용될 수 있다.According to the present invention, it was confirmed that homoharringtonine inhibits GRP78 (Glucose-Regulated Protein 78) expression, and that the anticancer effect of homoharringtonine is more significant in breast cancer with high GRP78 expression than in breast cancer with low GRP78 expression, thereby enabling the present invention to be usefully utilized as a composition for preventing, treating, or improving a disease caused by GRP78 overexpression; a method for preventing or treating the disease; or a reagent composition for inhibiting GRP78 expression.
도 1은 호모해링토닌(homoharringtonine; 이하 HHT라 함)이 ATPase 활성에 미치는 영향을 분석한 결과이다. *p<0.05, **p<0.01 및 ***p<0.001. Figure 1 shows the results of analyzing the effect of homoharringtonine (hereinafter referred to as HHT) on ATPase activity. *p<0.05, **p<0.01, and ***p<0.001.
도 2는 HHT가 GRP78(Glucose-Regulated Protein 78) 발현에 미치는 영향을 분석한 결과이다. *p<0.05, **p<0.01 및 ***p<0.001. Figure 2 shows the results of analyzing the effect of HHT on GRP78 (Glucose-Regulated Protein 78) expression. *p<0.05, **p<0.01, and ***p<0.001.
도 3A는 유방암 세포 종류별 GRP78 발현을 분석한 결과이고, 도 3B는 유방암 세포 종류별 HHT의 항암 효과를 분석한 결과이다. ****p<0.0001.Figure 3A shows the results of analyzing GRP78 expression by breast cancer cell type, and Figure 3B shows the results of analyzing the anticancer effect of HHT by breast cancer cell type. ****p<0.0001.
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은 호모해링토닌(homoharringtonine)을 유효성분으로 포함하는 GRP78(Glucose-Regulated Protein 78) 과발현에 의한 질환 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating a disease caused by GRP78 (Glucose-Regulated Protein 78) overexpression, which contains homoharringtonine as an active ingredient.
상기 호모해링토닌은 ATPase 활성을 억제할 수 있다.The above homoharringtonine can inhibit ATPase activity.
GRP78은 transmembrane ER 스트레스 센서를 비활성 형태로 결합하고 유지하며, ER 스트레스 발생 시, GRP78이 방출된다. 또한, ER 스트레스 inducer인 튜니카마이신(tunicamycin)은 잘못 접힌 단백질을 많이 생성함으로써 세포에서 ER 스트레스를 유도하여 GRP78의 단백질 발현을 증가시킨다. GRP78 binds and maintains the transmembrane ER stress sensor in an inactive form, and GRP78 is released when ER stress occurs. In addition, tunicamycin, an ER stress inducer, induces ER stress in cells by generating a large amount of misfolded proteins, thereby increasing the protein expression of GRP78.
상기 질환은 다발성 골수종, 폐암, 췌장암, 대장암, 유방암, 비인두암, 신장암, 교아종, 난소암, 간암, 담도암, 골육종, 유두갑상샘암, 위암, 식도암, 전립선암, 설암(tongue cancer) 및 윌름즈 종양(Wilms tumor)으로 이루어진 군에서 선택된 하나 이상일 수 있으나, 이에 한정되는 것은 아니다.The above disease may be one or more selected from the group consisting of, but is not limited to, multiple myeloma, lung cancer, pancreatic cancer, colon cancer, breast cancer, nasopharyngeal cancer, kidney cancer, glioblastoma, ovarian cancer, liver cancer, biliary tract cancer, osteosarcoma, papillary thyroid cancer, stomach cancer, esophageal cancer, prostate cancer, tongue cancer, and Wilms tumor.
본 발명의 약학 조성물은 당해 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다.The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains.
상기 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸 히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘, 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약학 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.The pharmaceutically acceptable carriers mentioned above are those commonly used in the preparation of formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.
본 발명에 있어서, 상기 약학 조성물에 포함되는 첨가제의 함량은 특별히 한정되는 것은 아니며 통상의 제제화에 사용되는 함량 범위 내에서 적절하게 조절될 수 있다.In the present invention, the content of the additive included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.
상기 약학 조성물은 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립, 정제, 크림, 젤, 패취, 분무제, 연고제, 경고제, 로션제, 리니멘트제, 파스타제 및 카타플라스마제로 이루어진 군에서 선택된 하나 이상의 피부 외용제 형태로 제형화될 수 있으나, 이에 한정되는 것은 아니다.The above pharmaceutical composition may be formulated in the form of one or more skin external preparations selected from the group consisting of injectable preparations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, pastes, lotions, liniments, pastes, and cataplasmas, but is not limited thereto.
본 발명의 약학 조성물은 제형화를 위해 추가로 있는 약학적으로 허용 가능한 담체 및 희석제를 포함할 수 있다. 상기 약학적으로 허용 가능한 담체 및 희석제는 전분, 당 및 만니톨과 같은 부형제, 칼슘 포스페이트 등과 같은 충전제 및 증량제, 카르복시메틸셀룰로오스, 히드록시프로필셀룰로오스 등과 같은 셀룰로오스 유도체, 젤라틴, 알긴산염, 폴리비닐 피롤리돈 등과 같은 결합제, 활석, 스테아린산 칼슘, 수소화 피마자유 및 폴리에틸렌글리콜과 같은 윤활제, 포비돈 및 크로스포비돈과 같은 붕해제, 폴리소르베이트, 세틸알코올, 글리세롤 등과 같은 계면활성제를 포함하나, 이에 한정되지 않는다. 상기 약학적으로 허용 가능한 담체 및 희석제는 대상체에게 생물학적 및 생리학적으로 친화적인 것일 수 있다. 희석제의 예로는 염수, 수용성 완충액, 용매 및/또는 분산제(dispersion media)를 들 수 있으나, 이에 제한되는 것은 아니다.The pharmaceutical composition of the present invention may further comprise pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include, but are not limited to, excipients such as starch, sugar and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose, binders such as gelatin, alginates and polyvinyl pyrrolidone, lubricants such as talc, calcium stearate, hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone and crospovidone, and surfactants such as polysorbates, cetyl alcohol, glycerol and the like. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents and/or dispersion media.
본 발명의 약학 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여(예를 들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있다. 경구 투여일 경우, 정제, 트로키제(troches), 로젠지(lozenge), 수용성 현탁액, 유성 현탁액, 조제 분말, 과립, 에멀젼, 하드 캡슐, 소프트 캡슐, 시럽, 엘릭시르제 등으로 제형화될 수 있다. 비경구 투여일 경우, 주사액, 좌제, 호흡기 흡입용 분말, 스프레이용 에어로졸제, 연고, 도포용 파우더, 오일, 크림 등으로 제형화 될 수 있다.The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. In the case of oral administration, it may be formulated as tablets, troches, lozenges, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, elixirs, etc. In the case of parenteral administration, it may be formulated as injections, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for application, oils, creams, etc.
본 발명의 약학 조성물의 투여량은 환자의 상태, 체중, 연령, 성별, 건강상태, 식이 체질 특이성, 제제의 성질, 질병의 정도, 조성물의 투여시간, 투여방법, 투여기간 또는 간격, 배설율 및 약물 형태에 따라 그 범위가 다양할 수 있으며, 이 분야 통상의 기술자에 의해 적절하게 선택될 수 있다. 예컨대, 약 0.1 내지 10,000mg/kg의 범위일 수 있으나 이제 제한되지 않으며, 하루 일회 내지 수회에 나누어 투여될 수 있다.The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition, weight, age, sex, health condition, dietary constitution, nature of the preparation, degree of disease, administration time of the composition, administration method, administration period or interval, excretion rate, and drug form, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg/kg, but is not limited thereto, and may be administered once a day or divided into several times.
상기 약학 조성물은 목적하는 방법에 따라 경구 투여되거나 비경구 투여(예를 들면, 정맥 내, 피하 내, 복강 내 또는 국소에 적용)될 수 있다. 본 발명의 약학 조성물의 약학적 유효량 및 유효 투여량은 약학 조성물의 제제화 방법, 투여 방식, 투여 시간, 투여 경로 등에 의해 다양해질 수 있으며, 당해 기술 분야에서 통상의 지식을 가진 자는 목적하는 치료에 효과적인 투여량을 용이하게 결정하고 처방할 수 있다. 본 발명의 약학 조성물의 투여는 하루에 1회 투여될 수 있고, 수회에 나누어 투여될 수도 있다.The pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. The pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method of the pharmaceutical composition, the administration method, the administration time, the administration route, etc., and a person having ordinary knowledge in the art can easily determine and prescribe an effective dosage for the intended treatment. The pharmaceutical composition of the present invention may be administered once a day or may be administered in several divided doses.
또한, 본 발명은 호모해링토닌(homoharringtonine)을 유효성분으로 포함하는 GRP78(Glucose-Regulated Protein 78) 과발현에 의한 질환 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or improving a disease caused by GRP78 (Glucose-Regulated Protein 78) overexpression, which contains homoharringtonine as an active ingredient.
본 발명은 통상적으로 이용되는 식품으로써 일반적으로 사용될 수 있다.The present invention can be generally used as a commonly used food.
본 발명의 식품 조성물은 건강기능식품으로서 사용될 수 있다. 상기 “건강기능식품”이라 함은 건강기능 식품에 관한 법률에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 의미하며, “기능성”이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다.The food composition of the present invention can be used as a health functional food. The above “health functional food” means a food manufactured and processed using raw materials or ingredients having functionality useful to the human body according to the Health Functional Food Act, and “functionality” means that it is consumed for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects.
상기 건강기능식품 조성물은 통상의 식품 첨가물을 포함할 수 있으며, 상기 “식품 첨가물”로서의 적합 여부는 다른 규정이 없는 한, 식품의약품안전처에 승인된 식품 첨가물 공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다.The above health functional food composition may contain conventional food additives, and its suitability as the above “food additive” is determined by the specifications and standards for the relevant item according to the general provisions and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.
상기 “식품 첨가물 공전”에 수재된 품목으로는 예를 들어, 케톤류, 글리신, 구연산칼륨, 니코틴산, 계피산 등의 화학적 합성물, 감색소, 감초추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물, L-글루타민산나트륨 제제, 면류첨가알칼리제, 보존료제제, 타르색소제제 등의 혼합제제류들을 들 수 있다.Items listed in the above “Food Additives Codex” include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular-weight pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents added to noodles, preservative preparations, and tar color preparations.
본 발명의 식품 조성물은 정제, 캡슐, 분말, 과립, 액상, 환 등의 형태로 제조 및 가공할 수 있다. 예를 들어, 캡슐 형태의 건강기능 식품 중 경질 캡슐제는 통상의 경질 캡슐에 본 발명에 따른 조성물을 부형제 등의 첨가제와 혼합 및 충진 하여 제조할 수 있으며, 연질 캡슐제는 본 발명에 따른 조성물을 부형제 등의 첨가제와 혼합하고 젤라틴 등 캡슐기제에 충진하여 제조할 수 있다. 상기 연질 캡슐제 는 필요에 따라 글리세린 또는 소르비톨 등의 가소제, 착색제, 보존제 등을 함유할 수 있다.The food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. For example, among health functional foods in the form of capsules, hard capsules can be manufactured by mixing and filling a composition according to the present invention with additives such as excipients into a conventional hard capsule, and soft capsules can be manufactured by mixing a composition according to the present invention with additives such as excipients and filling a capsule base such as gelatin. The soft capsules can contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., as necessary.
상기 부형제, 결합제, 붕해제, 활택제, 교미제, 착향제 등에 대한 용어 정의 는 당업계에 공지된 문헌에 기재된 것으로 그 기능 등이 동일 내지 유사한 것들을 포함한다. 상기 식품의 종류에는 특별한 제한이 없으며, 통상적인 의미에서의 건강 기능식품을 모두 포함한다.The definitions of terms for the above excipients, binders, disintegrants, lubricants, maturing agents, flavoring agents, etc. are those described in literature known in the art and include those having the same or similar functions. There is no particular limitation on the type of the above food, and all health functional foods in the conventional sense are included.
본 발명에서 용어 “예방”은 본 발명에 따른 조성물의 투여로 GRP78 과발현에 의한 질환을 억제 또는 지연시키는 모든 행위를 말한다. The term “prevention” in the present invention refers to any act of suppressing or delaying a disease caused by GRP78 overexpression by administering a composition according to the present invention.
본 발명에서 용어 “치료”는 본 발명에 따른 조성물의 투여로 GRP78 과발현에 의한 질환의 증세가 호전되거나 이롭게 변경하는 모든 행위를 말한다. The term “treatment” in the present invention refers to any act of improving or beneficially changing the symptoms of a disease caused by GRP78 overexpression by administering a composition according to the present invention.
본 발명에서 용어 “개선”은 본 발명에 따른 조성물의 투여로 GRP78 과발현에 의한 질환의 나쁜 상태를 좋게 하는 모든 행위를 말한다.The term “improvement” in the present invention refers to any act of improving the bad condition of a disease caused by GRP78 overexpression by administering a composition according to the present invention.
또한, 본 발명은 인간을 제외한 개체군에 있어서, 일반 환자군 대비 GRP78(Glucose-Regulated Protein 78) 발현이 높은 환자군에 호모해링토닌(homoharringtonine)을 처리하는 단계(제1단계)를 포함하는, GRP78 과발현에 의한 질환 예방 또는 치료 방법을 제공한다.In addition, the present invention provides a method for preventing or treating a disease caused by GRP78 overexpression, including a step (first step) of treating homoharringtonine in a patient group having a high expression of GRP78 (Glucose-Regulated Protein 78) compared to a general patient group in a population other than humans.
본 발명은 호모해링토닌(homoharringtonine)을 유효성분으로 포함하는 GRP78(Glucose-Regulated Protein 78) 발현 억제용 시약 조성물을 제공한다.The present invention provides a reagent composition for inhibiting GRP78 (Glucose-Regulated Protein 78) expression, which contains homoharringtonine as an active ingredient.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, in order to help understand the present invention, examples will be given and described in detail. However, the following examples are only intended to illustrate the content of the present invention, and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to a person having average knowledge in the art.
[[ 실험예Experimental example 1] 실험 준비1] Experiment preparation
1-1. 세포 배양1-1. Cell culture
인간유래 피부 섬유아세포(human dermal fibroblast cell; 이하 HDF 세포라 함)는 ATCC(Manassas, VA, USA)에서 구입하여 사용하였다. 세포를 10% FBS(fetal bovine serum)(GIBCO, NE, USA) 및 1×페니실린-스트렙토마이신(Hyclone, Logan, UT, USA)이 첨가된 DMEM(Dulbecco’s Modified Eagle’s Medium)(WelGENE, Gyeongsan-si, Korea)을 사용하여 37℃ 및 5% CO2 조건에서 배양하였다. 그 후, 세포에 HHT(100nM, TOCRIS, 영국)를 처리하여 2시간 동안 배양하고, 튜니카마이신(tunicamycin, 2μg/mL)을 처리한 후, 하룻밤 동안 배양하였다. Human dermal fibroblast cells (HDF cells) were purchased from ATCC (Manassas, VA, USA). The cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) (WelGENE, Gyeongsan-si, Korea) supplemented with 10% FBS (fetal bovine serum) (GIBCO, NE, USA) and 1× penicillin-streptomycin (Hyclone, Logan, UT, USA) at 37°C and 5% CO 2. The cells were then treated with HHT (100 nM, TOCRIS, UK) and cultured for 2 hours, then treated with tunicamycin (2 μg/mL), and cultured overnight.
삼중음성 유방암세포(MDA-MB-231 및 HCC-1937)는 ATCC(Manassas, VA, USA)에서 구입하여 사용하였다. 세포를 10% FBS(GIBCO, NE, USA) 및 1×페니실린-스트렙토마이신(GIBCO, NE, USA)이 첨가된 RPMI1640(Roswell Park Memorial Institute 1640)(WelGENE, Gyeongsan-si, Korea)을 사용하여 37℃ 및 5% CO2 조건에서 배양하였다.Triple-negative breast cancer cells (MDA-MB-231 and HCC-1937) were purchased from ATCC (Manassas, VA, USA). Cells were cultured in RPMI1640 (Roswell Park Memorial Institute 1640) (WelGENE, Gyeongsan-si, Korea) supplemented with 10% FBS (GIBCO, NE, USA) and 1× penicillin-streptomycin (GIBCO, NE, USA) at 37°C and 5% CO 2 .
1-2. 비만 동물모델1-2. Obese animal model
8주령 C57BL/6 수컷 마우스(KOATECH, Pyeongtaek-si, Korea)를 1주일 동안 사육환경에 적응시켰다. 실험군은 하기와 같이 설정하였다. 마우스를 정상식이군 및 고지방식이군으로 나누고, 고지방식이군은 총 칼로리의 60.3%가 지방인 식이(#TD.06414, R&D systems, Minneapolis, MN, USA)를 6주 동안 섭취시켰다. 그 후, 고지방식이군을 대조군 및 실험군으로 나누고, 대조군은 PBS를, 실험군은 HHT를 복강 내 투여하였다. 구체적으로, HHT 10mg을 DMSO(Dimethyl sulfoxide)에 10mM로 용해시키고, PBS(Phosphate-buffered saline) 3.9μl에 HHT 10mM 0.1μl를 넣어 희석하였다. PBS에 희석된 HHT를 마우스 체중 g당 4μl(HHT 0.545μg)씩 투여하였다. 따라서 마리당 0.545μg/g body weight/1회를 8주 동안 주 3회씩 복강 내 투여하였다(총 투여량 : 13.08μg/g body weight). 정상군에도 같은 용량의 DMSO가 희석된 PBS를 복강 내 투여하였다(총 투여량 : 96μl/g body weight). 마우스는 실험 종료 시까지 22±2℃의 실내온도와 12시간의 명암주기를 조절한 환경에서 사육하였고, 1주 간격으로 체중 및 식이 섭취량을 측정하였다. 실험 종료 후, 마우스를 Avertin 마취제로 마취시키고, 지방조직(adipose tissue)을 채취하여 분석 전까지 -80℃에서 보관하였다.Eight-week-old C57BL/6 male mice (KOATECH, Pyeongtaek-si, Korea) were acclimated to the breeding environment for 1 week. The experimental groups were set up as follows. The mice were divided into a normal diet group and a high-fat diet group, and the high-fat diet group was fed a diet in which 60.3% of the total calories were fat (#TD.06414, R&D systems, Minneapolis, MN, USA) for 6 weeks. After that, the high-fat diet group was divided into a control group and an experimental group, and the control group was administered PBS and the experimental group was administered HHT intraperitoneally. Specifically, 10 mg of HHT was dissolved in DMSO (Dimethyl sulfoxide) to 10 mM and diluted with 0.1 μl of 10 mM HHT in 3.9 μl of PBS (PBS). The HHT diluted in PBS was administered 4 μl (0.545 μg of HHT) per g of mouse body weight. Therefore, 0.545 μg/g body weight/time per mouse was administered intraperitoneally three times a week for 8 weeks (total administration: 13.08 μg/g body weight). The normal group was also administered intraperitoneally PBS containing the same amount of DMSO (total administration: 96 μl/g body weight). The mice were raised in an environment with a controlled room temperature of 22±2℃ and a 12-h light/dark cycle until the end of the experiment, and their body weight and food intake were measured at weekly intervals. After the end of the experiment, the mice were anesthetized with Avertin anesthesia, and their adipose tissue was collected and stored at -80℃ until analysis.
1) 정상군(Chow) : 정상식이 + PBS(96μl/g body weight) 투여1) Normal group (Chow): Normal diet + PBS (96μl/g body weight) administered
2) 대조군(HFD) : 고지방식이 + PBS(96μl/g body weight) 투여2) Control group (HFD): High-fat diet + PBS (96μl/g body weight) administration
3) 실험군(HFD+HHT) : 고지방식이 + HHT(13.08μg/g body weight) 투여3) Experimental group (HFD+HHT): High-fat diet + HHT (13.08μg/g body weight) administered
1-3. 노화 동물모델1-3. Aging animal model
18개월된 C57BL/6 마우스(KOATECH, Pyeongtaek-si, Korea)를 1주일 동안 사육환경에 적응시켰다. HHT는 상기 실시예 1-2와 동일한 방법으로 DMSO에 용해시키고, PBS로 희석하여 사용하였다. 실험군은 하기와 같이 설정하였다. 마우스를 대조군 및 실험군으로 나누고, 대조군은 PBS를, 실험군은 HHT(0.545μg/g body weight/1회)를 1주일에 3회, 총 19주 동안 복강 내 투여하였다(총 투여량 : 31.065μg/g body weight). 대조군은 PBS(4μl/g body weight/1회)를 1주일에 3회, 총 19주 동안 복강 내 투여하였다(총 투여량 : 228μg/g body weight). 마우스는 실험 종료 시까지 22±2℃의 실내온도와 12시간의 명암주기를 조절한 환경에서 사육하였고, 1주 간격으로 체중 및 식이 섭취량을 측정하였다. 실험 종료 후, 마우스를 Avertin 마취제로 마취시키고, 앞정강근(tibialis anterior muscle)을 채취하여 분석 전까지 -80℃에서 보관하였다.18-month-old C57BL/6 mice (KOATECH, Pyeongtaek-si, Korea) were acclimated to the breeding environment for 1 week. HHT was dissolved in DMSO in the same manner as in Example 1-2, and diluted with PBS for use. The experimental groups were set up as follows. The mice were divided into a control group and an experimental group. The control group was administered PBS and the experimental group was administered HHT (0.545 μg/g body weight/once) intraperitoneally three times a week for a total of 19 weeks (total dosage: 31.065 μg/g body weight). The control group was administered PBS (4 μl/g body weight/once) intraperitoneally three times a week for a total of 19 weeks (total dosage: 228 μg/g body weight). The mice were raised in an environment controlled at an indoor temperature of 22±2℃ and a 12-hour light/dark cycle until the end of the experiment, and their body weight and food intake were measured at weekly intervals. After the experiment, the mice were anesthetized with Avertin, and the tibialis anterior muscle was collected and stored at -80°C until analysis.
1) 대조군(PBS) : PBS(228μl/g body weight) 투여1) Control group (PBS): PBS (228μl/g body weight) administered
2) 실험군(HHT) : HHT(31.065μg/g body weight) 투여2) Experimental group (HHT): HHT (31.065μg/g body weight) administered
[[ 실험예Experimental example 2] 2] ATPaseATPase 활성 분석Active Analysis
HHT가 ATPase 활성에 미치는 영향을 확인하기 위해, 제조사의 지침에 따라 ATPase Activity Assay Kit(Sigma-Aldrich; Merck KGaA, Darmstadt, Germany)를 사용하여 ATPase 활성을 측정하였다. 40mM Tris, 80mM NaCl, 8mM MgAc2, 1mM EDTA 및 4mM ATP를 혼합하고(pH 7.5), GRP78 단백질(5μg); GRP78 단백질(5μg) + HHT(10μM); 또는 GRP78 단백질(5μg) + HA15(10μM, HTT 억제제, 양성대조군)를 혼합하여 반응 혼합물을 제조한 후, 실온에서 60분 동안 배양하였다. 그 후, 각 웰에 상기 kit에 포함된 시약[reagent(MAK113A)] 200μL를 첨가하고, 실온에서 추가로 10~30분 동안 배양하여 효소 반응을 종료한 후, 비색 생성물을 생성하였다. 상기 비색 생성물을 96 웰 플레이트로 옮기고, 마이크로플레이트 리더(Sunrise™, TECAN, Switzerland)를 이용하여 620nm에서의 인산염(Phosphate) 활성(흡광도)을 측정하였다. 인산염 표준물질을 사용하여 표준곡선을 설정하였다.To determine the effect of HHT on ATPase activity, ATPase activity was measured using an ATPase Activity Assay Kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) according to the manufacturer's instructions. 40 mM Tris, 80 mM NaCl, 8 mM MgAc 2 , 1 mM EDTA, and 4 mM ATP were mixed (pH 7.5), and GRP78 protein (5 μg); GRP78 protein (5 μg) + HHT (10 μM); or GRP78 protein (5 μg) + HA15 (10 μM, HTT inhibitor, positive control) were mixed to prepare a reaction mixture, and then incubated at room temperature for 60 min. After that, 200 μL of the reagent [reagent (MAK113A)] included in the kit was added to each well, and the enzyme reaction was completed by incubating for an additional 10 to 30 minutes at room temperature, and a colorimetric product was generated. The colorimetric product was transferred to a 96-well plate, and the phosphate activity (absorbance) at 620 nm was measured using a microplate reader (Sunrise™, TECAN, Switzerland). A standard curve was established using phosphate standards.
[[ 실험예Experimental example 3] 3] GRP78GRP78 발현 억제 활성 분석Expression inhibition activity assay
HHT가 GRP78(NCBI gene ID : 3309) 발현에 미치는 영향을 확인하기 위해, 웨스턴 블롯(Western blot)을 수행하였다. 상기 실험예 1-1에서 배양한 세포에서 배지를 걷어내고, DPBS(Dulbecco′s Phosphate Buffered Saline)(Gyeongsan-si, Korea)로 세척한 후, 각 세포를 수거하였다. 수거한 세포에 protease inhibitor가 포함된 lysis buffer[RIPA buffer : 25mM Tris-HCl(pH 7.6), 150mM NaCl, 1% NP-40, 1% sodium deoxycholate 및 0.1% SDS]로 용해시키고, 4℃에서 13,000rpm으로 원심분리하여 단백질을 추출하였다. 또한, 상기 실험예 1-2 및 1-3에서 채취하여 급속 냉동한 동물조직의 일정량에 protease inhibitor가 포함된 lysis buffer[150mM NaCl, 50mM HEPES, 50mM 플루오린화나트륨(NaF), 1mM 벤즈아마이드(benzamide), 1mM EGTA, 1mM EDTA, 1mM 디티오트레이톨(dithiothreitol), 1mM 오소바나데이트 나트륨(sodium orthovanadate), 1mM 페닐메틸설포닐 플루오라이드(phenylmethylsulfonyl fluoride), 1% NP-40, 10% 글리세롤(glycerol), 0.22% β-글리세로포스페이트(β-glycerophosphate)]를 넣고, 마이크로비드(microbeads)를 이용하여 균질화시킨 후, 4℃에서 13,000rpm으로 원심분리하여 단백질을 추출하였다.To confirm the effect of HHT on the expression of GRP78 (NCBI gene ID: 3309), Western blot was performed. The medium was removed from the cells cultured in Experimental Example 1-1, washed with Dulbecco's Phosphate Buffered Saline (DPBS) (Gyeongsan-si, Korea), and each cell was harvested. The harvested cells were lysed with lysis buffer containing protease inhibitor [RIPA buffer: 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS], and centrifuged at 13,000 rpm at 4°C to extract proteins. In addition, a lysis buffer containing protease inhibitor [150 mM NaCl, 50 mM HEPES, 50 mM sodium fluoride (NaF), 1 mM benzamide, 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1% NP-40, 10% glycerol, 0.22% β-glycerophosphate] was added to a certain amount of the animal tissues collected and rapidly frozen in the above Experimental Examples 1-2 and 1-3, homogenized using microbeads, and centrifuged at 13,000 rpm at 4°C. Protein was extracted.
상기 단백질들을 정량하고, SDS-PAGE를 수행한 후, 멤브레인(membrane)에 단백질을 이동시켰다. 그 후, 1차 항체 및 2차 항체를 차례로 반응시키고, ECL kit(Amersham Biosciences, NJ, USA)를 사용하여 화학발광(chemiluminescence)으로 확인하였다. 발현된 밴드(band)를 ImageJ(National Institute of Mental Health, Bethesda, Maryland, USA) 프로그램을 사용하여 정량하였다. 각 실험군의 단백질 수준은 각 실험군의 하우스키핑 단백질인 GAPDH 단백질 수준으로 보정하여 나타냈다.The above proteins were quantified, and after performing SDS-PAGE, the proteins were transferred to a membrane. After that, primary and secondary antibodies were reacted in sequence, and chemiluminescence was confirmed using an ECL kit (Amersham Biosciences, NJ, USA). The expressed bands were quantified using the ImageJ (National Institute of Mental Health, Bethesda, Maryland, USA) program. The protein levels of each experimental group were expressed by correcting them to the GAPDH protein level, which is a housekeeping protein of each experimental group.
[[ 실험예Experimental example 4] 통계 분석4] Statistical Analysis
통계적 분석은 GraphPad Prism 8 소프트웨어(GraphPad Software, San Diego, CA, USA)를 사용하여 수행하였다. 두 실험군 사이의 통계적 분석은 Student’s t-test를 이용하였고, 세 실험군 사이의 통계적인 분석은 One-way ANOVA 및 Tukey’s post-hoc test를 이용하였다. p<0.05를 통계적으로 유의한 것으로 간주하였다.Statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA). Statistical analysis between two experimental groups was performed using Student’s t-test, and statistical analysis between three experimental groups was performed using One-way ANOVA and Tukey’s post-hoc test. p<0.05 was considered statistically significant.
[[ 실시예Example 1] 1] ATPaseATPase 활성 분석Active Analysis
상기 실험예 2에 따라, 호모해링토닌이 ATPase 활성에 미치는 영향을 분석한 결과, 도 1과 같이, GRP78 단독 처리군(GRP78) 대비 HHT 처리군(GRP78+HHT) 및 양성대조군(GRP78+HA15)에서 ATPase 활성이 유의하게 감소하였다. 구체적으로, HHT는 ATPase 활성을 약 30% 감소시켰고, 양성대조군인 HA15는 ATPase 활성을 약 20% 감소시켰다. 상기 결과로부터, HHT는 ATPase 활성을 억제하는 것을 확인하였다.According to the above Experimental Example 2, the effect of homoharringtonine on ATPase activity was analyzed, and as shown in Fig. 1, ATPase activity was significantly reduced in the HHT treatment group (GRP78+HHT) and the positive control group (GRP78+HA15) compared to the GRP78 only treatment group (GRP78). Specifically, HHT reduced ATPase activity by approximately 30%, and the positive control group, HA15, reduced ATPase activity by approximately 20%. From the above results, it was confirmed that HHT inhibits ATPase activity.
[[ 실시예Example 2] 2] GRP78GRP78 발현 억제 활성 분석Expression inhibition activity assay
상기 실험예 3에 따라, HHT가 GRP78 발현에 미치는 영향을 분석한 결과, 도 2A와 같이, HDF 세포의 경우, 정상군(튜니카마이신 미처리군; Nor) 대비 대조군(튜니카마이신 단독 처리군; Con)에서 GRP78 단백질 발현이 유의하게 증가한 반면, 대조군 대비 HHT 처리군(HHT)에서 GRP78 단백질 발현이 유의하게 감소하였다. 또한, 도 2B와 같이, 비만 동물모델의 경우, 정상군(Chow) 대비 대조군(HFD)에서 GRP78 단백질 발현이 유의하게 증가한 반면, 대조군 대비 실험군(HHT)에서 GRP78 단백질 발현이 유의하게 감소하였다. 또한, 도 2C와 같이, 노화 동물모델의 경우, 대조군(PBS) 대비 실험군(HHT)에서 GRP78 단백질 발현이 유의하게 감소하였다. 상기 결과로부터, HHT는 GRP78 발현을 억제하는 것을 확인하였다.According to the above Experimental Example 3, the effect of HHT on GRP78 expression was analyzed and, as shown in Fig. 2A, in the case of HDF cells, GRP78 protein expression significantly increased in the control group (tunicamycin only treatment group; Con) compared to the normal group (tunicamycin untreated group; Nor), whereas GRP78 protein expression significantly decreased in the HHT treatment group (HHT) compared to the control group. In addition, as shown in Fig. 2B, in the case of the obese animal model, GRP78 protein expression significantly increased in the control group (HFD) compared to the normal group (Chow), whereas GRP78 protein expression significantly decreased in the experimental group (HHT) compared to the control group. In addition, as shown in Fig. 2C, in the case of the aging animal model, GRP78 protein expression significantly decreased in the experimental group (HHT) compared to the control group (PBS). From the above results, it was confirmed that HHT inhibits GRP78 expression.
[[ 실시예Example 3] 유방암 세포에서 항암 효과 분석3] Analysis of anticancer effects in breast cancer cells
3-1. 유방암 세포 종류별 3-1. By breast cancer cell type GRP78GRP78 발현 분석Expression analysis
유방암 세포 종류별 GRP78 발현을 확인하기 위해, 웨스턴 블롯(Western blot)을 수행하였다. 상기 실험예 1-1에서 배양한 삼중음성 유방암 세포를 수거하고, protease inhibitor가 포함된 lysis buffer[RIPA buffer : 25mM Tris-HCl(pH 7.6), 150mM NaCl, 1% NP-40, 1% sodium deoxycholate 및 0.1% SDS]로 용해시키고, 4℃에서 13,000rpm으로 원심분리하여 단백질을 추출하였다. 이후 상기 실험예 3과 동일한 방법으로 GRP78 발현을 확인하였다.To confirm GRP78 expression by breast cancer cell type, Western blot was performed. Triple-negative breast cancer cells cultured in Experimental Example 1-1 were harvested, lysed with lysis buffer containing protease inhibitor [RIPA buffer: 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS], and centrifuged at 13,000 rpm at 4°C to extract proteins. Thereafter, GRP78 expression was confirmed using the same method as Experimental Example 3.
그 결과, 도 3A와 같이, HCC-1937 세포 대비 MDA-MB-231 세포에서 GRP78 발현이 유의하게 높게 나타났다. 상기 결과로부터, 유방암 세포 종류별로 GRP78 발현 차이가 나타남을 확인하였다.As a result, as shown in Fig. 3A, GRP78 expression was significantly higher in MDA-MB-231 cells than in HCC-1937 cells. From the results above, it was confirmed that there was a difference in GRP78 expression depending on the breast cancer cell type.
3-2. 유방암 세포 종류별 3-2. By breast cancer cell type HHT의HHT's 항암 효과 분석Anticancer effect analysis
유방암 세포 종류별 HHT의 항암 효과를 확인하기 위해, 상기 실험예 1-1에서 배양한 삼중음성 유방암 세포에 HHT(100nM, TOCRIS, 영국)를 처리하여 3일 동안 배양하였다. 그 후, 상기 배양한 배양액 200μL에 WST 시약 10μL(EZ-Cytox, DoGenBio, seoul, Korea)을 처리하고, 2~3시간 30분간 반응시킨 후, 마이크로플레이트 리더(Sunrise™, TECAN, Switzerland)를 이용하여 450nm에서의 흡광도를 측정하여 세포 생존율을 확인하였다.To confirm the anticancer effect of HHT according to breast cancer cell type, triple-negative breast cancer cells cultured in Experimental Example 1-1 were treated with HHT (100 nM, TOCRIS, UK) and cultured for 3 days. Then, 10 μL of WST reagent (EZ-Cytox, DoGenBio, seoul, Korea) was treated to 200 μL of the cultured medium, and after reacting for 2 to 3 hours and 30 minutes, the cell viability was confirmed by measuring the absorbance at 450 nm using a microplate reader (Sunrise™, TECAN, Switzerland).
그 결과, 도 3B와 같이, GRP78 발현이 낮은 HCC-1937 세포 대비 GRP78 발현이 높은 MDA-MB-231 세포에서 HHT의 항암 효과(세포 성장 억제 효과)가 더 유의하게 나타났다. 구체적으로, HHT 처리군에서 MDA-MB-231 세포 및 HCC-1937 세포의 세포 생존율은 각각 약 20.5% 및 47.4%로 나타났다. 상기 결과로부터, HHT는 GRP78 발현이 낮은 유방암 대비 GRP78 발현이 높은 유방암에 대한 항암 효과가 더 우수한 것을 확인하였다.As a result, as shown in Fig. 3B, the anticancer effect (cell growth inhibition effect) of HHT was more significant in MDA-MB-231 cells with high GRP78 expression than in HCC-1937 cells with low GRP78 expression. Specifically, the cell viability rates of MDA-MB-231 cells and HCC-1937 cells in the HHT treatment group were approximately 20.5% and 47.4%, respectively. From the above results, it was confirmed that HHT had a more excellent anticancer effect on breast cancer with high GRP78 expression than on breast cancer with low GRP78 expression.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 즉, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다.While the specific parts of the present invention have been described in detail above, it is obvious to those skilled in the art that such specific description is merely a preferred embodiment and that the scope of the present invention is not limited thereby. In other words, the actual scope of the present invention is defined by the appended claims and their equivalents.
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