WO2020138560A1 - Composition pharmaceutique et aliment fonctionnel de santé pour prévenir ou traiter des cataractes - Google Patents

Composition pharmaceutique et aliment fonctionnel de santé pour prévenir ou traiter des cataractes Download PDF

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Publication number
WO2020138560A1
WO2020138560A1 PCT/KR2018/016832 KR2018016832W WO2020138560A1 WO 2020138560 A1 WO2020138560 A1 WO 2020138560A1 KR 2018016832 W KR2018016832 W KR 2018016832W WO 2020138560 A1 WO2020138560 A1 WO 2020138560A1
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WO
WIPO (PCT)
Prior art keywords
pharmaceutical composition
cataracts
dexamethasone
cataract
health functional
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PCT/KR2018/016832
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English (en)
Korean (ko)
Inventor
김성재
Original Assignee
경상대학교병원
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Priority to PCT/KR2018/016832 priority Critical patent/WO2020138560A1/fr
Publication of WO2020138560A1 publication Critical patent/WO2020138560A1/fr

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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 COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/12—Ophthalmic agents for cataracts

Definitions

  • the present invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment of cataracts.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a pharmaceutical composition for preventing or treating cataracts comprising a protein consisting of the amino acid sequence of SEQ ID NO: 1.
  • the steroid is dexamethasone (Dexamethasone), pharmaceutical composition.
  • the formulation of the pharmaceutical composition is an eye drop, the pharmaceutical composition.
  • a health functional food for cataract prevention or improvement comprising a protein consisting of the amino acid sequence of SEQ ID NO: 1.
  • the cataract is a steroid-induced cataract, health functional food.
  • 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.
  • 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.
  • FIG. 7 and 8 are diagrams showing a heating map as a result of RT 2 profiler PCR analysis.
  • FIGS 9 and 10 are diagrams showing the effect of NGF on cell viability in DEX-treated HLE-B3.
  • FIG. 11 and 12 are diagrams showing the effect of NGF on cell migration in DLE-treated HLE-B3.
  • FIG. 13 is a view showing the effect of NGF on EMT marker in DEX-treated HLE-B3.
  • FIGS 14 and 15 are diagrams showing the effect of NGF on downstream signals in DLE-treated HLE-B3.
  • compositions 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.
  • administration refers to the introduction of a predetermined substance to an individual in an appropriate manner
  • 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.
  • the pharmaceutical composition of the present invention may further include a composition having a known cataract prevention or treatment effect.
  • Taiwanese propolis TP
  • Mori folium 1 ⁇ 10 Euonymus alatus 1 ⁇ 10: Ginseng Radix 1
  • Water extracts ⁇ -crystallin charge masking agents, and the like, but are not limited thereto.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • carrier examples 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.
  • the health functional food of the present invention 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.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are usually used.
  • HLE-B3 Human lens epithelial B-3 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).
  • 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.
  • Movement analysis was performed using a specific wound assay chamber (ibidi GmbH, Kunststoff, 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.
  • Amplification was performed under the following conditions: 50° C., 2 min in 96 well plates using ViiATM 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.
  • 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).
  • each blot is fibronectin, ⁇ -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 ⁇ -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.
  • 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 ⁇ g of total RNA was converted to cDNA in a 10 ⁇ l reaction volume using RT2 First Strand Kit (Qiagen). cDNA was diluted in RT2 SYBR Green Mastermix (Qiagen) and a volume of 4,200 ⁇ l of distilled water. 10 ⁇ l was used for each primer set in the pathway-specific RT2 Profiler PCR Arrays (Qiagen) according to the manufacturer's protocol.
  • 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.
  • FIG. 1 is a heating map where red and green represent increases and decreases, respectively.
  • NGF was shown to decrease in all groups when compared to the control group, which was confirmed again by PCR.
  • expression of p-TrkA increased when 500ng/ml NGF in HLEC was treated (FIGS. 7 and 8 ).
  • fibronectin and alpha-SMA increased at a significantly reduced dexamethasone administration in dexamethasone and NGF treated groups (FIG. 13 ).

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Abstract

La présente invention concerne une composition pharmaceutique comprenant une protéine composée d'une séquence d'acides aminés SEQ ID NO : 1, cette composition pouvant prévenir ou traiter des cataractes, en particulier des cataractes induites par les stéroïdes, et un aliment fonctionnel de santé comprenant une protéine composée d'une séquence d'acides aminés SEQ ID NO : 1, cet aliment pouvant prévenir ou atténuer des cataractes, en particulier des cataractes induites par les stéroïdes, et ainsi la présente invention est utile dans le traitement, l'atténuation des symptômes ou similaire de patients souffrant de cataractes.
PCT/KR2018/016832 2018-12-28 2018-12-28 Composition pharmaceutique et aliment fonctionnel de santé pour prévenir ou traiter des cataractes WO2020138560A1 (fr)

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PCT/KR2018/016832 WO2020138560A1 (fr) 2018-12-28 2018-12-28 Composition pharmaceutique et aliment fonctionnel de santé pour prévenir ou traiter des cataractes

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4039269A1 (fr) * 2021-02-05 2022-08-10 Dompe' Farmaceutici S.P.A. Isoforme ngf à utiliser dans le traitement des pathologies oculaires

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100298282A1 (en) * 2006-01-19 2010-11-25 Alan Geoffrey Roach Treatment of Ocular Conditions and the Side-Effects of Glucocorticoids
JP2018008922A (ja) * 2015-08-04 2018-01-18 わかもと製薬株式会社 ステロイド白内障の予防及び治療

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100298282A1 (en) * 2006-01-19 2010-11-25 Alan Geoffrey Roach Treatment of Ocular Conditions and the Side-Effects of Glucocorticoids
JP2018008922A (ja) * 2015-08-04 2018-01-18 わかもと製薬株式会社 ステロイド白内障の予防及び治療

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE NCBI, GenBank 26 July 2016 (2016-07-26), "beta-nerve growth factor", XP055722665, Database accession no. BAA90437.1 *
GHINELLI, E.: "Nerve growth factor (NGF) and lenses: effects of NGF in an in vitro rat model of cataract", GRAEFE'S ARCHIVE FOR CLINICAL AND EXPERIMENTAL OPHTHALMOLOGY, 2003, pages 845 - 851, XP055722663 *
PETERSEN, A.: "Effects of dexamethasone on human lens epithelial cells in culture", MOLECULAR VISION, 21 July 2008 (2008-07-21), pages 1344 - 1352, XP055722667 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4039269A1 (fr) * 2021-02-05 2022-08-10 Dompe' Farmaceutici S.P.A. Isoforme ngf à utiliser dans le traitement des pathologies oculaires
WO2022167607A1 (fr) * 2021-02-05 2022-08-11 Dompe' Farmaceutici Spa Isoforme ngf destiné à être utilisé dans le traitement de pathologies oculaires

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