Pharmaceutical composition for preventing or treating diseases caused by GRP78 overexpression, comprising homoharringtonine as active ingredient

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating diseases caused by glucose-regulated protein 78 (GRP78) overexpression, the composition comprising homoharringtonine as an active ingredient. Homoharringtonine was found to inhibit GRP78 expression, and the anticancer effect of homoharringtonine was found to be more significant in breast cancer having high GRP78 expression than breast cancer having low GRP78 expression. Thus, homoharringtonine can be effectively used in: a composition for preventing, treating, or alleviating diseases caused by GRP78 overexpression; a method for preventing or treating said diseases; or a reagent composition for inhibiting GRP78 expression.

Description

Pharmaceutical composition for preventing or treating diseases caused by GRP78 overexpression, containing homoharringtonine as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for preventing or treating a disease caused by overexpression of GRP78 (Glucose-Regulated Protein 78), which contains homoharringtonine as an active ingredient.

[0002] GRP78 (Glucose-Regulated Protein 78) is a 654-amino acid protein 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 its ATPase activity. HA15, a commercially available GRP78 inhibitor, is known to bind to GRP78 and inhibit its ATPase activity.

[0003] Meanwhile, homoharringtonine, a single natural compound isolated from Cephalotaxushainanensis, is commercially used as an adjuvant treatment for chronic myeloid leukemia. Its mechanism of action is known to inhibit protein production by binding to the 80S ribosome in eukaryotic cells and inhibiting chain elongation. However, the relationship between homoharringtonine and GRP78 has not yet been reported.

[0004] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating a disease caused by overexpression of GRP78 (Glucose-Regulated Protein 78).

[0005] Another object of the present invention is to provide a health functional food composition for preventing or improving diseases caused by overexpression of GRP78 (Glucose-Regulated Protein 78).

[0006] Another object of the present invention is to provide a method for preventing or treating a disease caused by GRP78 overexpression, which comprises 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.

[0007] Another object of the present invention is to provide a reagent composition for inhibiting GRP78 (Glucose-Regulated Protein 78) expression.

[0008] To achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating a disease caused by overexpression of GRP78 (Glucose-Regulated Protein 78), which contains homoharringtonine as an active ingredient.

[0009] In addition, the present invention provides a health functional food composition for preventing or improving a disease caused by overexpression of GRP78 (Glucose-Regulated Protein 78), which contains homoharringtonine as an active ingredient.

[0010] In addition, the present invention provides a method for preventing or treating a disease caused by GRP78 overexpression, which comprises 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.

[0011] In addition, the present invention provides a reagent composition for inhibiting GRP78 (Glucose-Regulated Protein 78) expression, which contains homoharringtonine as an active ingredient.

[0012] According to the present invention, it was confirmed that homoharringtonine suppresses 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, and thus the present invention can 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 suppressing GRP78 expression.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] Hereinafter, the present invention will be described in more detail.

[0017]

[0018] The present invention provides a pharmaceutical composition for preventing or treating a disease caused by overexpression of GRP78 (Glucose-Regulated Protein 78), which contains homoharringtonine as an active ingredient.

[0019] The above homoharringtonine can inhibit ATPase activity.

[0020] GRP78 binds and maintains the transmembrane ER stress sensor in an inactive form, and GRP78 is released upon ER stress. Furthermore, tunicamycin, an ER stress inducer, induces ER stress in cells by increasing the production of misfolded proteins, thereby increasing GRP78 protein expression.

[0021] 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.

[0022] 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.

[0023] The pharmaceutically acceptable carriers mentioned above are those commonly used in 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.

[0024] 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.

[0025] The above pharmaceutical composition may be formulated in the form of one or more external skin preparations selected from the group consisting of injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, and cataplasmas, but is not limited thereto.

[0026] 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, and glycerol. 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.

[0027] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., 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.

[0028] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition, weight, age, sex, health status, dietary constitution, nature of the formulation, severity of the 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 or several times a day in divided doses.

[0029] 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, administration method, administration time, administration route, etc. of the pharmaceutical composition. A person skilled 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 divided into several doses.

[0030]

[0031] In addition, the present invention provides a health functional food composition for preventing or improving a disease caused by overexpression of GRP78 (Glucose-Regulated Protein 78), which contains homoharringtonine as an active ingredient.

[0032] The present invention can be generally used as a commonly used food.

[0033] The food composition of the present invention can be used as a health functional food. The term "health functional food" refers to a food manufactured and processed using raw materials or ingredients with functional properties useful to the human body as defined in the Health Functional Food Act. "Functionality" refers to ingestion for the purpose of obtaining beneficial effects for health purposes, such as regulating nutrients for the structure and function of the human body or physiological effects.

[0034] 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 in accordance with the general provisions and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.

[0035] Items listed in the above “Food Additives Code” 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 for noodles, preservative preparations, and tar color preparations.

[0036] 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 capsule form, 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 may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., as necessary.

[0037] The definitions of terms for the above excipients, binders, disintegrants, lubricants, flavoring agents, etc. are described in documents known in the art and include those with the same or similar functions. There is no particular limitation on the type of food, and all health functional foods in the conventional sense are included.

[0038] In the present invention, the term “prevention” refers to any act of suppressing or delaying a disease caused by GRP78 overexpression by administering a composition according to the present invention.

[0039] In the present invention, the term “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.

[0040] In the present invention, the term “improvement” 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.

[0041]

[0042] In addition, the present invention provides a method for preventing or treating a disease caused by GRP78 overexpression, which comprises 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.

[0043]

[0044] The present invention provides a reagent composition for inhibiting GRP78 (Glucose-Regulated Protein 78) expression, which contains homoharringtonine as an active ingredient.

[0045] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0046]

[0047] [Experimental Example 1] Experimental Preparation

[0048] 1-1. Cell culture

[0049] Human dermal fibroblast cells (HDF cells) were purchased from ATCC (Manassas, VA, USA). Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (WelGENE, Gyeongsan-si, Korea) supplemented with 10% fetal bovine serum (FBS) (GIBCO, NE, USA) and 1× penicillin-streptomycin (Hyclone, Logan, UT, USA) at 37°C and 5% CO2. Cells were then treated with HHT (100 nM, TOCRIS, UK) and cultured for 2 hours, followed by treatment with tunicamycin (2 μg / mL) and cultured overnight.

[0050] 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% CO2.

[0051]

[0052] 1-2. Obesity animal model

[0053] Eight-week-old C57BL / 6 male mice (KOATECH, Pyeongtaek-si, Korea) were acclimated to the breeding environment for one 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 containing 60.3% of total calories as fat (#TD.06414, R&D systems, Minneapolis, MN, USA) for 6 weeks. Thereafter, the high-fat diet group was divided into a control group and an experimental group, and the control group was administered PBS intraperitoneally, and the experimental group was administered HHT. Specifically, 10 mg of HHT was dissolved in DMSO (Dimethyl sulfoxide) to a 10 mM solution and diluted with 0.1 μl of 10 mM HHT in 3.9 μl of phosphate-buffered saline (PBS). The HHT diluted in PBS was administered at a dose of 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 with PBS containing the same amount of DMSO (total administration: 96 μl / g body weight). The mice were housed in an environment with a controlled room 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 end of the experiment, the mice were anesthetized with Avertin anesthesia, and the adipose tissue was collected and stored at -80℃ until analysis.

[0054] 1) Normal group (Chow): Normal diet + PBS (96 μl / g body weight) administered

[0055] 2) Control group (HFD): High-fat diet + PBS (96 μl / g body weight) administered

[0056] 3) Experimental group (HFD+HHT): High-fat diet + HHT (13.08 μg / g body weight) administered

[0057]

[0058] 1-3. Animal models of aging

[0059] 18-month-old C57BL / 6 mice (KOATECH, Pyeongtaek-si, Korea) were acclimated to the breeding environment for one 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 dose: 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 dose: 228 μg / g body weight). The mice were raised in an environment controlled at room temperature of 22±2℃ and a 12-hour light / dark cycle until the end of the experiment, and body weight and food intake were measured at weekly intervals. After the experiment, the mice were anesthetized with Avertin anesthetic, and the tibialis anterior muscle was collected and stored at -80°C until analysis.

[0060] 1) Control group (PBS): PBS (228 μl / g body weight) administered

[0061] 2) Experimental group (HHT): HHT (31.065 μg / g body weight) administered

[0062]

[0063] [Experimental Example 2] ATPase Activity Analysis

[0064] 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 MgAc2, 1 mM EDTA, and 4 mM ATP (pH 7.5) were mixed, and reaction mixtures were prepared by mixing GRP78 protein (5 μg); GRP78 protein (5 μg) + HHT (10 μM); or GRP78 protein (5 μg) + HA15 (10 μM, HTT inhibitor, positive control), and incubated at room temperature for 60 min. Afterwards, 200 μL of the reagent (MAK113A) included in the kit was added to each well and incubated at room temperature for an additional 10 to 30 minutes to complete the enzymatic reaction and generate a colorimetric product. 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.

[0065]

[0066] [Experimental Example 3] Analysis of GRP78 expression inhibition activity

[0067] To determine the effect of HHT on the expression of GRP78 (NCBI gene ID: 3309), Western blotting 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 inhibitors [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.

[0068] The above proteins were quantified, subjected to SDS-PAGE, and transferred to a membrane. Primary and secondary antibodies were then reacted sequentially, and the protein was confirmed by chemiluminescence using an ECL kit (Amersham Biosciences, NJ, USA). The expressed bands were quantified using the ImageJ program (National Institute of Mental Health, Bethesda, Maryland, USA). The protein levels in each experimental group were expressed by correcting them to the levels of GAPDH, the housekeeping protein in each experimental group.

[0069]

[0070] [Experimental Example 4] Statistical Analysis

[0071] 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 between three experimental groups, one-way ANOVA and Tukey's post-hoc test were used. A p<0.05 value was considered statistically significant.

[0072]

[0073] [Example 1] ATPase activity analysis

[0074] 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 treatment group alone (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.

[0075]

[0076] [Example 2] Analysis of GRP78 expression inhibition activity

[0077] 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 treated group; Con) compared to the normal group (tunicamycin-untreated group; Nor), whereas GRP78 protein expression significantly decreased in the HHT-treated 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.

[0078]

[0079] [Example 3] Analysis of anticancer effects in breast cancer cells

[0080] 3-1. Analysis of GRP78 expression by breast cancer cell type

[0081] 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 proteins were extracted by centrifugation at 13,000 rpm at 4°C. Thereafter, GRP78 expression was confirmed using the same method as Experimental Example 3.

[0082] As a result, as shown in Figure 3A, GRP78 expression was significantly higher in MDA-MB-231 cells compared to HCC-1937 cells. From the above results, it was confirmed that GRP78 expression differed depending on the breast cancer cell type.

[0083]

[0084] 3-2. Analysis of the anticancer effects of HHT by breast cancer cell type

[0085] To confirm the anticancer effect of HHT on different breast cancer cell types, 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 added to 200 μL of the culture medium, and the cells were allowed to react for 2 to 3 hours and 30 minutes. Then, the cell viability was confirmed by measuring the absorbance at 450 nm using a microplate reader (Sunrise™, TECAN, Switzerland).

[0086] 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 of MDA-MB-231 cells and HCC-1937 cells in the HHT treatment group was approximately 20.5% and 47.4%, respectively. From the above results, it was confirmed that HHT had a better anticancer effect on breast cancer with high GRP78 expression than on breast cancer with low GRP78 expression.

[0087]

[0088] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for preventing or treating a disease caused by overexpression of GRP78 (Glucose-Regulated Protein 78), containing homoharringtonine as an active ingredient.

2. A composition according to claim 1, characterized in that the homoharringtonine inhibits ATPase activity.

3. A composition according to claim 1, characterized in that the disease is at least one selected from the group consisting of multiple myeloma, lung cancer, pancreatic cancer, colon cancer, breast cancer, nasopharyngeal cancer, renal cancer, glioblastoma, ovarian cancer, liver cancer, biliary tract cancer, osteosarcoma, papillary thyroid cancer, stomach cancer, esophageal cancer, prostate cancer, tongue cancer, and Wilms tumor.

4. A health functional food composition containing homoharringtonine as an active ingredient for preventing or improving diseases caused by overexpression of GRP78 (Glucose-Regulated Protein 78).

5. 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.

6. A composition for inhibiting the expression of GRP78 (Glucose-Regulated Protein 78) containing homoharringtonine as an active ingredient.

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