WO2020138560A1 - Pharmaceutical composition and health functional food for preventing or treating cataracts - Google Patents

Abstract

A pharmaceutical composition comprising a protein composed of an amino acid sequence of SEQ ID NO: 1, of the present invention, can prevent or treat cataracts, particularly steroid-induced cataracts, and a health functional food comprising a protein composed of an amino acid sequence of SEQ ID NO: 1, of the present invention, can prevent or alleviate cataracts, particularly steroid-induced cataracts, and thus the present invention is useful in the treatment, alleviation or the like of patients suffering from cataracts.

Description

Pharmaceutical composition and health functional food for cataract prevention or treatment

The present invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment of cataracts.

When a person reaches a certain age, the lens of the eye (the organ corresponding to the lens of the camera) hardens and becomes cloudy, making it difficult to pass light, which gradually blurs the field of view. In particular, when the oxidative stress caused by free radicals acts on the eyes, the protein of the lens may be oxidized to induce cataracts, and the older the older, the higher the probability of cataract induction because the defense against oxidative stress such as free radicals decreases. The initial incidence rate of cataracts is about 60% in the 60s and 80% in the 70s, and most of them occur in the 80s.

The causes of cataracts are various, such as oxidative stress, protein aggregation, and steroid induction, and when cataracts occur, they may appear cloudy or fogged, or symptoms such as blindness, glare, and overlapping objects may occur. Once cataracts develop, use of eye drops to suppress progression, or surgical removal of cataract-induced lens and insertion of an artificial lens is a treatment currently being performed. In the case of the surgical treatment method, a lot of progress has been made in the last 10 years, but the effect of the method for treating cataracts by non-surgical methods such as eye drops such as eye drops or oral treatments is still weak.

Accordingly, there is a need for a cataract treatment that can be provided as an eye drop or a pharmaceutical composition that is a non-surgical method.

The present invention aims to provide a pharmaceutical composition for preventing or treating cataracts comprising a protein consisting of the amino acid sequence of SEQ ID NO: 1.

In addition, an object of the present invention is to provide a health functional food for cataract prevention or improvement comprising a protein consisting of the amino acid sequence of SEQ ID NO: 1.

1. A pharmaceutical composition for preventing or treating cataracts comprising a protein consisting of the amino acid sequence of SEQ ID NO: 1.

2. The method of 1 above, wherein the cataract is a steroid-induced cataract, pharmaceutical composition.

3. In the above 1, the steroid is dexamethasone (Dexamethasone), pharmaceutical composition.

4. In the above 1, the formulation of the pharmaceutical composition is an eye drop, the pharmaceutical composition.

5. A health functional food for cataract prevention or improvement comprising a protein consisting of the amino acid sequence of SEQ ID NO: 1.

6. In the above 5, the cataract is a steroid-induced cataract, health functional food.

7. In the above 5, the steroid is dexamethasone (Dexamethasone), health functional food.

The pharmaceutical composition comprising the protein consisting of the amino acid sequence of SEQ ID NO: 1 of the present invention can prevent or treat cataracts, particularly steroid-induced cataracts.

In addition, a health functional food comprising a protein consisting of the amino acid sequence of SEQ ID NO: 1 of the present invention can prevent or improve cataracts, especially steroid-induced cataracts.

1 and 2 is a diagram showing the effect of dexamethasone on the cell viability in HLE-B3.

3 and 4 are diagrams showing the effect of dexamethasone on wound healing analysis in HLE-B3.

5 and 6 are diagrams showing the effect of dexamethasone on EMT in HLE-B3.

7 and 8 are diagrams showing a heating map as a result of RT ² profiler PCR analysis.

9 and 10 are diagrams showing the effect of NGF on cell viability in DEX-treated HLE-B3.

11 and 12 are diagrams showing the effect of NGF on cell migration in DLE-treated HLE-B3.

13 is a view showing the effect of NGF on EMT marker in DEX-treated HLE-B3.

14 and 15 are diagrams showing the effect of NGF on downstream signals in DLE-treated HLE-B3.

Hereinafter, the present invention will be described in detail.

It relates to a pharmaceutical composition for preventing or treating cataracts comprising a protein consisting of the amino acid sequence of SEQ ID NO: 1.

The protein consisting of the amino acid sequence of SEQ ID NO: 1 is a human-derived nerve growth factor (NGF) protein.

The protein consisting of the amino acid sequence of SEQ ID NO: 1 included in the present invention may be derived from nature, or may be synthesized using a known protein synthesis method.

The protein consisting of the amino acid sequence of SEQ ID NO: 1 may be a product obtained by culturing peptides, plant-derived tissue or cell extracts, microorganisms (eg, bacteria or fungi, and especially yeast).

The pharmaceutical composition according to the present invention may include an active ingredient alone or may be provided as a pharmaceutical composition including one or more pharmaceutically acceptable carriers, excipients, or diluents.

"Pharmaceutically acceptable" in the present invention means that it exhibits properties that are not toxic to cells or humans exposed to the composition.

The pharmaceutical composition of the present invention may be provided by mixing with a composition for preventing or treating cataracts known in the art. That is, the pharmaceutical composition of the present invention can be administered in parallel with a known composition having a cataract prevention or treatment effect.

In the present invention, "administration" refers to the introduction of a predetermined substance to an individual in an appropriate manner, and "individual" refers to all living things, such as rats, mice, and livestock, including humans capable of carrying cataracts. As a specific example, it may be a mammal, including a human.

If necessary, the pharmaceutical composition of the present invention may further include a composition having a known cataract prevention or treatment effect.

As a composition for preventing or treating cataracts, the extract of Taiwanese propolis (TP), Mori folium 1~10: Euonymus alatus 1~10: Ginseng Radix 1, a combination of herbal medicines Water extracts, γ-crystallin charge masking agents, and the like, but are not limited thereto.

In the present invention, the route of administration of the pharmaceutical composition is, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, Topical, sublingual, ocular or rectal.

The composition of the present invention may be administered orally or parenterally, and when parenterally administered, it is preferable to select an external injection or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection injection method. , More preferably, it may be a method of injecting into the eye in the form of an eye drop when applying the skin, but is not limited thereto.

The preferred dosage of the composition of the present invention depends on the patient's condition and body weight, the degree of disease, the drug form, the route and duration of administration, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition is preferably administered at 0.01 to 1000 mg/kg/day, preferably 0.1 to 500 mg/kg/day, but is not limited thereto. The administration may be administered once a day, or may be divided into several times. The above dosage does not limit the scope of the present invention in any way.

In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient in the extract, for example, starch, calcium carbonate, sucrose. Or it is prepared by mixing lactose, gelatin, and the like. Also, lubricants such as magnesium stearate and talc are used in addition to simple excipients. Liquid preparations for oral use include suspensions, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, can be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, etc. may be used, and a known diluent or excipient is preferably used in the form of eye drops. Can be used.

The cause of the cataract may be, for example, steroid, radiation, infection, excessive intake of sodium, and the like, but the cataract may be a steroid-induced cataract, but is not limited thereto.

In addition, the steroid is not limited as long as it is a steroid that can induce cataracts, but may be, for example, Dexamethasone.

The pharmaceutical composition comprising a protein consisting of the amino acid sequence of SEQ ID NO: 1 may increase the cell viability due to steroids, and may reduce cell mobility due to steroids, but is not limited thereto.

In addition, the pharmaceutical composition comprising a protein consisting of the amino acid sequence of SEQ ID NO: 1 can increase the expression of EMT marker fibronectin and alpha-SMA reduced by steroids, and NGF, AKT and p38 MAPK reduced by steroids And may increase signal transmission, but is not limited thereto.

In addition, the present invention relates to a health functional food for cataract prevention or improvement comprising a protein consisting of the amino acid sequence of SEQ ID NO: 1.

The cause of the cataract may be, for example, steroid, radiation, infection, excessive intake of sodium, and the like, but the cataract may be a steroid-induced cataract, but is not limited thereto.

In addition, the steroid is not limited as long as it is a steroid that can induce cataracts, but may be, for example, Dexamethasone.

The health functional food of the present invention may be formulated as one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, further comprising one or more of carriers, diluents, excipients, and additives. Foods to which the extract of the present invention can be added include various foods, powders, granules, tablets, capsules, syrups, drinks, gums, teas, vitamin complexes, and health functional foods.

Additives that may be further included in the present invention include natural carbohydrates, flavoring agents, nutrients, vitamins, minerals (electrolytes), flavoring agents (synthetic flavoring agents, natural flavoring agents, etc.), coloring agents, fillers (cheese, chocolate, etc.), Factic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, antioxidants, glycerin, alcohols, carbonizing agents and one or more components selected from the group consisting of flesh can be used. .

Examples of the natural carbohydrates described above include monosaccharides, such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, etc.; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agents (taumatine, stevia extract (for example, rebaudioside A, glycyrrhizine, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and neutralizing agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid And salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonic acid used in carbonated beverages, and the like. In addition, the composition according to the present invention may contain natural fruit juice and pulp for the production of vegetable beverages. These ingredients can be used independently or in combination.

Specific examples of the carrier, excipients, diluents and additives include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium phosphate, calcium Silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate And it is preferred that one or more selected from the group consisting of mineral oil is used.

When formulating the health functional food of the present invention, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are usually used.

Hereinafter, examples will be described in detail to specifically describe the present invention.

Example

1. Experimental methods and materials

(1) Cell culture and treatment

Human lens epithelial B-3 (HLE-B3) cells, an HLEC cell line immortalized by SV-40 viral transformation, were purchased from the American Type Culture Collection (ATCC; Rockville, MD). HLE-B3 cells were Dulbecco's modified Eagle medium supplemented with 37°C, 5% CO ₂ /95% O ₂ , supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco BRL, Grand Island, NY). Dulbecco's modified Eagle's medium (DMEM). For HLE-B3 cell treatment, dexamethasone (Dex) was purchased from Sigma-Aldrich (St. Louis, MO). Cells are left untreated or stimulated with Dex (0.1 mg/ml) for 24, 48 or 72 hours while human recombinant NGF (hrNGF) (R&D Systems, Minneapolis, MN) (SEQ ID NO: 1) is added Or not added.

(2) Cell viability analysis

HLE-B3 cells were seeded in 24-well plates at a concentration of 5 x 10 ⁴ cells per well and incubated for 24 hours. The next day, cells were treated at various concentrations of Dex (0, 0.01, 0.1, 1 mg/ml) for various time intervals (24, 48, 72 hours). Cell viability was measured using Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). Briefly, 10 ml per well of CCK-8 solution was added and incubated for 1 hour at 37°C in a humidified 5% CO ₂ atmosphere. The amount of formazan dye produced by cellular dehydrogenase activity was determined by measuring absorbance at 450 nm using a microplate reader (Molecular Devices, Sunnyvale, CA). During exposure at various concentrations of Dex, cells were observed using an inverted microscope (Nikon, Tokyo, Japan) to confirm the phenotypic difference between Dex treated and untreated cells.

(3) Cell migration assay

Movement analysis was performed using a specific wound assay chamber (ibidi GmbH, Munich, Germany). HLE-B3 cells were trypsinized and resuspended in DMEM-10% FBS. Cells were seeded with 4 x 10 ⁴ cells in each well of the insert and attached overnight. The next day, culture inserts were removed and optical microscopy images were obtained (3 per condition). Cells were cultured during transfer assay under Dex treated and untreated conditions, after which images were acquired 8 hours later using an inverted microscope (Nikon) equipped with an image capture system. Images were analyzed using automated image analysis software (Nikon NIS Elements software). For NGF experiments, NGF (R&D Systems) diluted in PBS was applied at 500 ng/mL.

(4) Quantitative RT-PCR analysis

Total RNA was extracted from each differentiating periosteal cell at the specified time, and the first strand cDNA was first-strand cDNA synthesis kit 7 (Applied Biosystems, Framingham, MA, USA) according to the manufacturer's instructions. It was generated using any of the hexamer (hexamer) primers provided in. All primers and probes (GAPDH Cat # Hs02758991; Fibronectin Cat # Hs0159976; α-SMA Cat # Hs 00426835) were obtained commercially (TaqMan® Gene Expression Assay, Applied Biosystems, Framingham, MA, USA). TaqMan® Gene Expression Master Mix, Applied Biosytems, Framingham, MA, USA). Amplification was performed under the following conditions: 50° C., 2 min in 96 well plates using ViiA™ 7 Real-Time PCR System (Applied Biosystems); 95° C., 10 minutes; 94°C, 15 seconds to 40 cycles; And 60° C., 1 minute. GAPDH was used as an internal control. All experiments were repeated three times.

(5) Western blot analysis

Cells were lysed in RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA) supplemented with PMSF, protease inhibitor cocktail, and sodium orthovanadate. Cells were sonicated and centrifuged for 10 minutes at 12,000 g at 4° C. to remove insoluble debris. Protein concentration in cell lysates was determined with the BCA Protein Assay Kit (Pierce, Rockford, IL). Total cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in 10% polyacrylamide gel and transferred to a nitrocellulose membrane (Millipore, Bedford, MA). After blocking with 5% nonfat dry milk, each blot is fibronectin, α-SMA, NGF (Abcam, Cambridge, MA), E-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA), Akt, phospho-Akt, Erk, phospho-Erk, Jnk, phospho-Jnk, p38, phospho-p38, Trk-A, phopho-Trk-A, (Cell Signaling Technology, Inc., Beverly, MA ) And β-actin (Sigma, St. Louis, MO) were incubated with primary antibodies, and then horseradish peroxidase-conjugated anti-rabbit immunoglobulin ) IgG or mouse IgG (Cell Signaling Technology, Inc.). Antibody binding was detected using a Supersignal Chemiluminescent Substrate (Pierce). Images were obtained with a ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA), and densitometry was performed with ImageJ software.

(6) Reversed transcription polymerase chain reaction array

Total RNA was purified from HLE-B3 cells using an RNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. Residual genomic DNA contaminants were removed using an RNase-Free DNase set (Qiagen). 5 μg of total RNA was converted to cDNA in a 10 μl reaction volume using RT² First Strand Kit (Qiagen). cDNA was diluted in RT² SYBR Green Mastermix (Qiagen) and a volume of 4,200 μl of distilled water. 10 μl was used for each primer set in the pathway-specific RT² Profiler PCR Arrays (Qiagen) according to the manufacturer's protocol. Quantitative RNA expression analysis of 84 genes related to growth factor is RT² The profiles were analyzed simultaneously using a PCR array (catalog # PAHS-041ZA; Qiagen). The amplification reaction was performed on an Applied Biosystems ViiA7 Real-Time PCR System (System) using automated baseline and threshold cycle detection. The threshold cycle number for each group of 84 genes is used by RT² Profiler PCR Array Data Analysis web-based software (SABiosciences Corporation, Frederick, MD, USA). Therefore, it was standardized with four built-in housekeeping gene controls.

(7) Statistical analysis

Quantifiable results were presented as the mean±standard deviation (SD) of at least 3 independent experiments. Statistical significance was determined by students' t-test. P values less than 0.05 were considered statistically significant.

2. Experimental results

(1) Effect of dexamethasone on cell viability in HLEC

Treatment of DEX at different doses and times in lens epithelial cells showed no difference between 0.01 and 0.1 mg/ml in the control groups, 24, 48 and 72 hours, but cell viability was dramatically reduced at 1 mg/ml DEX concentration (Figure 1). And 2).

(2) Effect of dexamethasone on wound healing analysis in HLEC

Cell migration analysis of 8 hour cultured HLECs administered with 0.01, 0.1 and 1 mg/ml DEX was increased by a concentration dependent manner. However, at a concentration of 1 mg/ml DEX, cell migration was significantly reduced compared to the control group (FIGS. 3 and 4 ).

(3) Effect of dexamethasone on EMT in HLEC

Both fibronectin and alpha-SMA as EMT markers increased significantly in the groups treated with 0.01 and 0.1 mg/ml dexamethasone after 24, 48 and 72, but E-cadherin was significantly reduced in both concentrations compared to the control group (Figure 5 and 6).

(4) RT ² Profiler PCR Array (RT ² result of profiler PCR array)

We divided the 0.1 mg dexamethasone treatment group into groups 1, 2 and 3 treated for 24, 48 and 72 hours, respectively, and compared to the control (Table 1). Figure 1 is a heating map where red and green represent increases and decreases, respectively. Of these, NGF was shown to decrease in all groups when compared to the control group, which was confirmed again by PCR. In addition, expression of p-TrkA increased when 500ng/ml NGF in HLEC was treated (FIGS. 7 and 8 ).

	Overexpression	Low expression
Group 1 (24 hours)	FIGF, IL12B, SLCO1A2	BMP5, NGF , FGF1, CSF2, IL11, HBEGF, NRG1, LIF, IL18, IGF1, INHBA, GDNF, IL1B, FGF5, BDNF, DKK1, PTN, NRG2
Group 2 (48 hours)	FGF6, FGF9, FGF19, CSPG5, CECR1, FIGF, LEFTY1, NODAL	BMP5, FGF1, HBEGF, IL11, NGF , NRG1, CSF2, INHBA, GDNF, LIF, DKK1, FGF5, MSTN, BDNF, OSGIN1, IL18, IGF1, PGF, IL1B
Group (72 hours)	FGF9, BMP6, FGF19, THPO, CLC, IL3, CSPG5	FGF1, HBEGF, BMP5, IL11, NRG1, CSF2, NGF , GDNF, INHBA, LIF, FGF5, DKK1, IL4, OSGIN1, PPC, IL1B, GDF11, JAG2, PTN, MSTN, PGF, GPI, BDNF, FGF2, IL18, BMP1, FGF7, ACTB, GAPDH, LTBP4, AMH, TGFB1

(5) Effect of NGF on cell viability in DEX treated HLEC

Compared to the 0.1 mg dexamethasone treated group, there was little change in cell viability in the NGF alone treated group. However, the survival rate was slightly increased in the group treated with NGF and dexamethasone, but was not statistically significant (FIGS. 9 and 10 ).

(6) Effect of NGF on cell migration in DEX treated HLEC

In the NGF and dexamethasone treated groups we found that increased cell mobility with dexamethasone treatment was significantly reduced by the level of the control or NGF treated groups (FIGS. 11 and 12 ).

(7) Effect of NGF on EMT marker expression in DEX treated HLEC

The expression of fibronectin and alpha-SMA increased at a significantly reduced dexamethasone administration in dexamethasone and NGF treated groups (FIG. 13 ).

(8) Effect of NGF on downstream signaling in DEX-treated HLEC

Among the downstream signals associated with NGF, AKT and p38 MAPK, it was reduced in the dexamethasone treatment group and increased when NGF was treated simultaneously (FIGS. 14 and 15 ).

Claims (7)

1. A pharmaceutical composition for preventing or treating cataracts comprising a protein consisting of the amino acid sequence of SEQ ID NO: 1.
2. The pharmaceutical composition of claim 1, wherein the cataract is a steroid-induced cataract.
3. The method according to claim 1, The steroid is dexamethasone (Dexamethasone), pharmaceutical composition.
4. The pharmaceutical composition of claim 1, wherein the formulation of the pharmaceutical composition is an eye drop.
5. A health functional food for cataract prevention or improvement comprising a protein consisting of the amino acid sequence of SEQ ID NO: 1.
6. The dietary supplement of claim 5, wherein the cataract is a steroid-induced cataract.
7. The method according to claim 5, The steroid is dexamethasone (Dexamethasone), health functional food.

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