WO2024029904A1 - Antioxidant composition including hapln1 and method for antioxidation in cells using same - Google Patents
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Classifications
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- 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
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
Definitions
- the present invention discloses an antioxidant composition for cells containing hyaluronic acid and proteoglycan link protein 1 (HAPLN1) or a gene encoding the same as an active ingredient, and a method for antioxidantizing cells using the composition. According to the present invention, by preventing and suppressing the oxidation function of cells, the occurrence or progression of various diseases caused by intervention of the oxidation function of cells is fundamentally suppressed, suggesting the possibility of preventing or treating the diseases.
- HPLN1 hyaluronic acid and proteoglycan link protein 1
- Reactive Oxygen Species is a chemical that can be converted into a stable water (H 2 O) molecule when an oxygen (O 2 ) molecule receives one electron and is reduced. This refers to high-energy, unstable oxygen generated during the process.
- Superoxide radicals (O 2- ), hydrogen peroxide (H 2 O 2 ), and hydroxy radicals (HO ⁇ ) are representative active oxygen radicals, which are internal factors caused by breathing and inflammation, which are essential for maintaining the living body. It is caused by external factors such as ultraviolet rays, radiation, smoking, environmental pollution, stress, and overeating.
- the human body is originally equipped with an antioxidant system that uses enzymes such as superoxide dismutase (SOD), hydrogen peroxide decomposition enzyme (Catalase), glutathione peroxidase, and glutathione reductase to remove these active oxygen radicals and protect cells.
- SOD superoxide dismutase
- Catalase hydrogen peroxide decomposition enzyme
- glutathione peroxidase glutathione reductase
- glutathione reductase glutathione reductase
- the present inventors conducted an in-depth study on hyaluronic acid and proteoglycan link protein 1 (HAPLN1) or the gene encoding it, and found that when various cells were treated with a composition containing them as active ingredients, abnormal oxidation of cells occurred.
- the present invention was completed by discovering surprising functions that can prevent or inhibit the action and normalize it.
- Patent Document 1 discloses HAPLN1 polypeptide as one of a wide range of individual factors secreted by bone marrow stromal cells (MSC, Marrow Stromal Cells), and when administered to an inflammatory disease subject, it can weaken the characteristics of the disease. It suggests that there is. However, nothing has been said about the antioxidant activity of cells.
- Non-Patent Document 1 is a paper on "Genetic Rescue of Chondrodysplasia and the Perinatal Lethal Effect of Cartilage Link Protein Deficiency", which uses genetic engineering. Mice in which the cartilage link protein (Crtl1) gene is destroyed show cartilage dysplasia and the mice die before and after birth. However, if a cartilage-specific link protein is introduced and overexpressed in Crtl1 null mice, abnormal cartilage formation is prevented and the mortality rate before and after birth is reduced. and the fact that Crtl1 transcript and its protein were found in all organs from fetuses to adults. However, even here, nothing is mentioned about the antioxidant effect on cells.
- Patent Document 1 US Published Patent US 2013/0052198
- Non-patent Document 1 Journal of Biological Chemistry.2003.Vol.278, No40.pp39214-29223
- the present invention provides an antioxidant composition containing hyaluronic acid and proteoglycan link protein 1 (HAPLN1) or a gene encoding the same as an active ingredient, and a method for antioxidantizing cells using the composition, thereby inhibiting the oxidation of cells.
- HPLN1 hyaluronic acid and proteoglycan link protein 1
- a reagent composition containing hyaluronic acid and proteoglycan link protein 1 (HAPLN1) or the gene encoding it as an active ingredient it can be used in research on the disease to identify the mechanism of the disease and treat related diseases.
- the aim is to contribute to the development of compositions for the prevention or treatment of.
- the present invention provides the following aspects of the invention.
- One aspect of the present invention relates to an antioxidant composition
- an antioxidant composition comprising hyaluronic acid and proteoglycan link protein 1 (HAPLN1) or a gene encoding the same as an active ingredient.
- HPLN1 proteoglycan link protein 1
- Another aspect of the present invention relates to an antioxidant composition in which the protein of the composition has more than 80% sequence identity to the amino acid sequence of SEQ ID NO: 1.
- Another aspect of the present invention relates to an antioxidant composition in which the nucleic acid related to the gene in the composition is contained in an expression vector.
- Another aspect of the present invention relates to an antioxidant composition in which the composition prevents or inhibits oxidation of cells.
- Another aspect of the present invention relates to an antioxidant composition in which the composition suppresses excessive generation of active oxygen in cells or reduces active oxygen already generated.
- Another aspect of the present invention relates to an antioxidant composition wherein the cells on which the composition acts are selected from the group consisting of fibroblasts, epithelial cells, and endothelial cells.
- one aspect of the present invention relates to an antioxidant composition in which the cells on which the composition acts are cells in an in vitro state.
- one aspect of the present invention relates to a composition in which a single dose of the composition to cells is 0.1 ng/ml to 1000 ng/ml,
- Another aspect of the present invention relates to an antioxidant composition that is included as a main or auxiliary active ingredient in a composition for preventing or inhibiting oxidation of cells.
- another aspect of the present invention is to treat cells with a composition containing hyaluronic acid and proteoglycan link protein 1 (HAPLN1) or the gene encoding it as an active ingredient, thereby promoting cell oxidation. It relates to methods of preventing or suppressing.
- HPLN1 hyaluronic acid and proteoglycan link protein 1
- an additional aspect of the present invention relates to a kit for preventing or inhibiting oxidation of cells, comprising the composition according to any one of items [1] to [9] and the processing instructions of item [10].
- an additional aspect of the present invention relates to an experimental reagent composition related to preventing or inhibiting oxidation of cells, comprising the composition according to any one of items [1] to [9].
- the occurrence or progression of various diseases caused by excessive oxidation of cells is fundamentally suppressed by preventing and inhibiting the oxidation of cells, thereby preventing or treating the diseases. It dramatically increases the possibility.
- the reagent composition of the present invention can be used in research on various diseases related to oxidation to help identify the mechanism of the disease and develop a composition for preventing or treating the disease.
- Figure 1 shows normal human skin fibroblasts treated with IL-1 ⁇ (10 ng/ml) to induce excessive production of reactive oxygen species (ROS) for 2 hours, and the composition of the present invention (rhHAPLN1) was administered at a concentration of 0 and 2.5, respectively.
- ROS reactive oxygen species
- A This is a graph showing changes in ROS levels by measuring the distribution of fluorescence generated by ROS in cells using FACS (BD FACSLyric TM , BD Biosciences).
- B This is a graph showing the relative intensity of DCF-DA for each content of the composition of the present invention as a multiple.
- C This is a table showing the average values of ROS levels per sample after performing three experiments.
- Figure 2 shows normal human skin fibroblasts treated with the composition of the present invention (rhHAPLN1) at 0, 2.5, 5, and 10 ng/ml under normal conditions and conditions that induce excessive production of reactive oxygen species (ROS), respectively.
- ROS reactive oxygen species
- Figure 3 shows normal human lung fibroblasts treated with no treatment, with only 5 ng/ml of the composition of the present invention (rhHAPLN1), and with IL-1 ⁇ (10 ng/ml) without the composition of the present invention.
- ROS reactive oxygen species
- Figure 4 shows the composition of the present invention (rhHAPLN1) on normal human lung fibroblasts under normal conditions (marked with “ ⁇ ”) and conditions inducing excessive production of reactive oxygen species (ROS) (marked with " ⁇ ”).
- ROS reactive oxygen species
- Figure 5 shows a sample of human corneal epithelial cells without any treatment (normal osmolality 314 mOsM), and samples in which generation of reactive oxygen species (ROS) was induced by preparing and treating 450 mOsM medium with 5M NaCl according to the present invention.
- This is an experimental result showing the ability to suppress active oxygen when the composition (rhHAPLN1) was treated with 0, 5, 10, 20, 50, and 100 ng/ml, respectively, and incubated for 24 hours.
- This is a graph showing the relative fluorescence intensity of DCF-DA due to active oxygen for each content of the composition of the present invention using Multi-Mode Reader (485/528nm, Synergy
- Figure 6 shows 0, 10, and 100 ng of the composition of the present invention (rhHAPLN1) to normal human corneal epithelial cells under normal conditions (314 mOsM) and conditions inducing excessive production of reactive oxygen species (ROS) (450 mOsM), respectively.
- ROS reactive oxygen species
- Photo showing the expression levels of antioxidant-related proteins expressed by cells in the sample such as NRF2 (Nuclear Factor erythroid-2-related factor 2), GPx1 (Glutathione peroxidase 1), and SOD1 (Superoxide dismutase 1), using SDS-PAGE and immunodetection methods.
- NRF2 Nuclear Factor erythroid-2-related factor 2
- GPx1 Glutathione peroxidase 1
- SOD1 Superoxide dismutase 1
- Figure 7 shows experimental results showing the ROS blocking performance of the composition of the present invention (rhHAPLN1) when reactive oxygen species (ROS) is directly introduced from the outside of the cell to human lung microvascular endothelial cells.
- H 2 O 2 250 ⁇ M was added to the sample to which no composition of the present invention (rhHAPLN1) was added and to the sample to which 30 ng/ml of the composition of the present invention (rhHAPLN1) was added, and H 2 O 2 increased over time.
- Figure 8a is an experimental result showing changes in the molecular level of antioxidant protein-related proteins when ROS is blocked by the composition of the present invention (rhHAPLN1) when reactive oxygen species (ROS) are introduced directly from the outside of the cell. 15 after adding H 2 O 2 (250 ⁇ M) to human lung microvascular endothelial cells to a sample to which no composition of the present invention (rhHAPLN1) was added and to a sample to which 30 ng/ml of the composition of the present invention (rhHAPLN1) was added.
- This is a photograph showing the expression level of various protein markers (pAkt, NRF2, pVEGFR2(Y1175), and VEGFR2) that respond to ROS over time, that is, indicators of antioxidant activity, using Western blot.
- Figure 8b shows H 2 O 2 (250 ⁇ M) for the sample to which no composition of the present invention (rhHAPLN1) was added and to the sample to which 30 ng/ml of the composition of the present invention (rhHAPLN1) was added under the experimental conditions of Figure 8a.
- This is a Western blot photo showing the expression level of VEGFR2, a protein marker that responds to ROS 2 hours after application, that is, an indicator of antioxidant activity.
- Figure 9 is an experimental result showing the change in cell proliferation rate by the composition of the present invention (rhHAPLN1) according to the dosage of the composition of the present invention when reactive oxygen species (ROS) are introduced directly from the outside of the cell.
- the composition of the present invention (rhHAPLN1) was administered to human lung microvascular endothelial cells at 0, 3, 10, and 30
- H 2 O 2 (1mM) was added, and the proliferation rate of cells was evaluated using Cell Counting kit-8 (Dojindo) at 24 hours (A) and 48 hours (B), and the graph (The control group is a sample to which neither the composition of the present invention nor H 2 O 2 was added).
- rhHAPLN1 is an abbreviation for recombinant human HAPLN1 and represents recombinant human HAPLN1.
- DCF-DA Assay means that DCF-DA (Dichlorofluorescein Diacetate) diffuses through the cell membrane and is hydrolyzed by intracellular esterase to become non-fluorescent DCF-H, This is a widely used analysis method to confirm the generation of ROS, using the principle that when ROS is present, it is oxidized very quickly to highly fluorescent DCF.
- DCF-DA Dichlorofluorescein Diacetate
- DCF-H is an abbreviation for 2,7-dichlorodihydrofluorescein.
- IL interleukin-1 ⁇
- NF ⁇ B Nuclear Factor kappa B
- ROS inflammatory response transcription factor
- NEF2 is an abbreviation for Nuclear Factor erythroid-2-related Factor 2, which is a representative transcription factor that affects the expression of antioxidant-related genes, and is found in the human body, such as the liver, lungs, digestive organs, brain, and skin. It is a protein distributed in major tissues.
- GPx1 is an abbreviation for Glutathione peroxidase 1, which is one of the most important antioxidant enzymes that reduces hydrogen peroxide to water.
- SOD1 is an abbreviation for Superoxide dismutase 1, which is an antioxidant enzyme present in the cytoplasm that catalyzes the reaction of converting superoxide anion into hydrogen peroxide.
- Akt refers to Protein Kinase B, a type of serine threonine kinase, and comes into direct contact with the pleckstrin-homology (PH) of synthesized PIP3, and phosphorylation occurs at the Thr308 and Ser473 sites, It binds to the cell plasma membrane and is activated. It is a factor that suppresses or promotes the production of reactive oxygen species depending on the signal transduction conditions, but it is understood that it mainly acts to activate NRF2 when ROS is excessive.
- PH pleckstrin-homology
- VEGFR2 is an abbreviation for Vascular Endothelial Growth Factor Receptor 2, which is a factor related to proliferation, survival, and angiogenesis in vascular endothelial cells, and is reduced by oxidative stress. do.
- One aspect of the present invention relates to an antioxidant composition
- an antioxidant composition comprising hyaluronic acid and proteoglycan link protein 1 (HAPLN1) or a gene encoding the same as an active ingredient.
- HPLN1 proteoglycan link protein 1
- the present invention may be the human recombinant HAPLN1 protein represented by SEQ ID NO: 1 in the composition of the present invention.
- the HAPLN1 protein of the present invention is 80%, preferably 85%, more preferably 90%, even more preferably 95%, and most preferably 80% of the amino acid sequence of SEQ ID NO: 1 as long as it maintains the antioxidant function. It may be a protein with 100% sequence identity.
- the nucleic acid related to the gene encoding the HAPLN1 protein in the composition of the present invention may be included in the composition by being included in an expression vector.
- the composition of the present invention exhibits the effect of preventing or suppressing the oxidation action of various cells, and the effect of preventing or suppressing the oxidation action of such cells is caused by excessive generation of active oxygen in the cell. It not only includes the effect of suppressing or reducing active oxygen that has already been generated, but also includes the effect of blocking active oxygen flowing in from the outside before and/or after the influx.
- the cells on which the composition of the present invention acts are not particularly limited, but are preferably fibroblasts, epithelial cells, and endothelial cells, and can be used in various situations such as in vivo, in vitro, ex vivo, and in situ. It includes, but is preferably in vitro.
- the composition of the present invention has a single dose to cells of 0.1 ng/ml to 1000 ng/ml, preferably 1 ng/ml to 500 ng/ml, more preferably 2.0 ng/ml. to 100 ng/ml, even more preferably 2.5 ng/ml to 50 ng/ml, and most preferably 3.0 ng/ml to 30 ng/ml.
- the composition of the present invention may be included as a main or auxiliary active ingredient in a composition for preventing or inhibiting oxidation of cells.
- preventing or inhibiting oxidation of cells by treating cells with a composition containing hyaluronic acid and proteoglycan link protein 1 (HAPLN1) or a gene encoding the same as an active ingredient. It's about how to do it.
- HPLN1 hyaluronic acid and proteoglycan link protein 1
- kits for preventing or inhibiting oxidation of cells including the composition of the present invention and processing instructions.
- a reagent composition for experiments related to preventing or inhibiting oxidation of cells comprising the composition of the present invention.
- the present invention relates to the use of the composition of the present invention for use in the production of a pharmaceutical composition for preventing or treating diseases caused by cellular oxidation.
- the pharmaceutical composition may generally include a molecule and a pharmaceutically acceptable carrier.
- pharmaceutically acceptable carrier includes saline solutions, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc. compatible with pharmaceutical administration. Supplementary active compounds may also be included in the composition.
- the pharmaceutical composition of the present invention may further include pharmaceutical additives selected from the group consisting of pharmaceutically acceptable carriers, diluents, binders, disintegrants, lubricants, and any combinations thereof.
- the pharmaceutical composition may be formulated to be compatible with the intended route of administration.
- the composition of the present invention may be in a dosage form selected from the group consisting of eye drops, ointments, tablets, pills, capsules, troches, inhalants, injections, patches, and suppositories.
- routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Preferably it is for parenteral administration.
- Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following ingredients: sterile diluents such as water for injection, saline, fixative oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; Antibacterial agents such as benzyl alcohol or methyl paraben; Antioxidants such as ascorbic acid or sodium bisulfite; Chelating agents such as ethylenediaminetetraacetic acid; Buffers such as acetate, citrate or phosphate and tonicity-adjusting agents such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be placed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.
- compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
- Carriers suitable for intravenous administration include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS).
- PBS phosphate buffered saline
- the composition must be sterile and fluid enough to facilitate injection. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and molds.
- the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof.
- suitable fluidity can be maintained by the use of a coating such as lecithin, maintenance of the required particle size in case of dispersion, and use of a surfactant.
- Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.
- isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, in the composition.
- Prolonged absorption of injectable compositions can be facilitated by including in the composition agents that delay absorption, such as aluminum monostearate and gelatin.
- Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
- Dispersions are generally prepared by incorporating the active compound into a sterile vehicle containing the basic dispersion medium and the other required ingredients listed above.
- the preferred preparation methods are vacuum drying and freeze drying, which yield a powder of the active ingredient and any additional ingredients desired from a previously sterile filtered solution.
- Oral compositions generally include an inert diluent or edible carrier.
- the active compounds may be incorporated with excipients and used in the form of tablets, troches or capsules, for example gelatin capsules.
- Oral compositions can also be prepared using fluid carriers for use as oral rinses. Pharmaceutically suitable binders and/or adjuvant substances may be included as part of the composition. Tablets, pills, capsules, troches, etc.
- a binder such as microcrystalline cellulose, gum tragacanth, or gelatin
- Excipients such as starch or lactose, disintegrants such as alginic acid, Primogel or corn starch
- Lubricants such as magnesium stearate or steroids
- Glidants such as colloidal silicon dioxide
- Sweeteners such as sucrose or saccharin
- a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
- the compound is delivered in the form of an aerosol spray from a pressure vessel or dispenser containing a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.
- a suitable propellant eg, a gas such as carbon dioxide, or a nebulizer.
- Systemic administration can also be achieved by transmucosal or transdermal means.
- a penetrating agent suitable for the barrier to be penetrated is used in the formulation.
- penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
- Transmucosal administration can be accomplished using a nasal spray or suppository.
- the active compounds are formulated as ointments, salves, gels or creams, as is generally known in the art.
- the compounds may also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
- suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
- retention enemas for rectal delivery.
- the dosage of these compounds is preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity. Dosages may vary within this range depending on the dosage form used and the route of administration used.
- the therapeutically effective amount (i.e., effective dosage) of the composition of the present invention will vary depending on the circumstances of the selected patient. For example, a single dose ranging from about 1 pg to 1000 mg may be administered; In some embodiments, 10, 30, 100, or 1000 pg, or 10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 ⁇ g, or 10, 30, 100, or 1000 mg may be administered. there is.
- the pharmaceutical dose is between 1 ng/ml and 100 ⁇ g/ml, preferably between 3 ng/ml and 50 ⁇ g/ml, more preferably between 5 ng/ml and 500 ng/ml, particularly preferably between 10 ng/ml and 200 ng/ml.
- the composition can be administered.
- the pharmaceutical composition can be administered from once a day to once a week, including once every other day.
- the skilled technician will recognize that certain factors may affect the dosage and timing required to effectively treat an individual, including the severity of the disease or disorder, prior treatment, the individual's general health and/or age, and other pre-existing conditions. Including, but not limited to, disease.
- treating an individual with a therapeutically effective amount of a molecule of the invention may include a single treatment or, preferably, may include a series of treatments.
- the composition of the present invention has a dosage of 5 mg/kg/week to 500 mg/kg/week, for example, 5 mg/kg/week, 10 mg/kg/week, and 15 mg/kg/week, depending on the patient's situation. , 20 mg/kg/week, 25 mg/kg/week, 30 mg/kg/week, 35 mg/kg/week, 40 mg/kg/week, 45 mg/kg/week, 50 mg/kg/week, 55mg/kg/week, 60mg/kg/week, 65mg/kg/week, 70mg/kg/week, 75mg/kg/week, 80mg/kg/week, 85mg/kg/week, 90mg/kg/week, 95mg/ kg/wk, 100mg/kg/wk, 150mg/kg/wk, 200mg/kg/wk, 250mg/kg/wk, 300mg/kg/wk, 350mg/kg/wk, 400mg/kg/wk, 450mg/kg/ week and
- the dosage of the bifunctional molecule according to the invention is 10 mg/kg/week to 200 mg/kg/week, 20 mg/kg/week to 150 mg/kg/week, or 25 mg/kg/week. It ranges from week to 100 mg/kg/week.
- the compositions of the invention can be administered for a period of 2 weeks to 6 months, e.g., 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks. Administered 1x weekly for 13 weeks, 26 weeks, 6 months, 8 months, 10 months, or more than 1 year.
- the compositions of the invention are administered 2x per week. In another embodiment, the compositions of the invention are administered every other week.
- composition of the present invention can also be formulated as a pharmaceutical composition containing a pharmacologically effective amount of HAPLN1 protein molecule and a pharmaceutically acceptable carrier.
- Pharmacologically or therapeutically effective amount means an amount effective to produce the intended pharmacological, therapeutic or prophylactic result.
- the phrases “pharmacologically effective amount” and “therapeutically effective amount” or simply “effective amount” refer to the amount of a bifunctional molecule that is effective to produce the intended pharmacological, therapeutic or prophylactic result. For example, if a given clinical treatment is considered effective when it reduces a measurable parameter associated with a disease or disorder by at least 20%, then a therapeutically effective dose of a drug to treat that disease or disorder would reduce that parameter by at least 20%. This is the amount needed to reduce it.
- compositions of the invention may be administered by parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, respiratory (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration. Administration may be by any means known in the art.
- the pharmaceutical composition is administered by intravenous or parenteral infusion or injection.
- suitable dosage units of the molecule range from 0.001 to 0.25 mg per kg of body weight of the recipient per day, or from 0.01 to 20 micrograms per kg of body weight per day, or from 0.01 to 10 micrograms per kg of body weight per day, or in the range of 0.10 to 5 micrograms per kilogram of body weight per day, or in the range of 0.1 to 2.5 micrograms per kilogram of body weight per day.
- a pharmaceutical composition containing the molecule can be administered once daily.
- the therapeutic agent may also be administered in dosage units comprising 2, 3, 4, 5, 6 or more subdoses administered at appropriate intervals throughout the day.
- Dosage units can also be formulated as single doses over several days, for example, using conventional sustained release formulations that provide sustained and consistent release of the molecule over a period of several days. Sustained release formulations are well known in the art.
- the dosage unit comprises a corresponding multiple of the daily dosage.
- the pharmaceutical composition may be included in a kit, container, pack, or dispenser along with administration instructions.
- treatment means treating, curing, alleviating, alleviating, altering, relieving, ameliorating, ameliorating, or affecting a disease or disorder, symptoms of a disease or disorder, or predisposition to a disease. It is defined as the application or administration of a therapeutic agent (e.g., a molecule of the invention) to a patient, or the application or administration of a therapeutic agent to an isolated tissue or cell line, for the purpose of treating the patient with a disease or disorder, symptoms of the disease or disorder, Or have a predisposition to disease or disability.
- a therapeutic agent e.g., a molecule of the invention
- one aspect of the present invention relates to the use of a composition containing hyaluronic acid and proteoglycan link protein 1 (HAPLN1) as active ingredients to prevent or inhibit oxidation of cells.
- HPLN1 hyaluronic acid and proteoglycan link protein 1
- HPLN1 hyaluronan and proteoglycan link protein 1
- a CHO-K1 cell line producing recombinant human HAPLN1 protein was created by inserting a vector containing a polynucleotide encoding the human HAPLN1 protein of amino acid sequence number 1 into CHO-K1 cells.
- amino acid sequence number 1 is as follows:
- MCB Master Cell Bank
- the MCB was subcultured and inoculated into Thermo's Hyperforma SUB 250 L bioreactor at a concentration of 0.40 ⁇ 0.05 ⁇ 10 6 cells/mL and fed-batch culture was performed.
- the basic medium used was 22.36 g ActiProTM medium + 0.5846 g glutamine + 10.00 g HT Supplement (Thermo Fisher Scientific) + 4.29 g 10 N NaOH + 1.80 g NaHCO 3 .
- the culture temperature was set at 36.5°C
- dissolved oxygen (DO) was set at 40.0%
- pH was set at 7.00 ⁇ 0.20. 1 M Sodium Carbonate Monohydrate was used as a pH adjustment solution.
- Recombinant human HAPLN1 (rhHAPLN1) protein was recovered from cells cultured as above (Harvest and Clarification), Ultrafiltration/Diafiltration 1 (UF/DF1), Anion Exchange Chromatography, S/D (Solvent/Detergent) ) Virus inactivation, cation exchange chromatography, Mixed-mode Chromatography (MMC), hydrophobic interaction chromatography, Ultrafiltration/Diafiltration 2 (UF/DF2) and intermediate depth filtration ( It was separated and purified through processes such as Intermediate Depth Filtration (Int. DF).
- the composition of the present invention was used.
- compositions of the invention comprising HAPLN1 protein
- the HAPLN1 protein purified in Preparation Example 1 was stabilized in 20 mM acetic acid buffer, 8% (w/v) sucrose, 0.04% (w/v) PS80, pH 5.0 to produce this product containing the HAPLN1 protein itself as the main active ingredient.
- the composition of the invention was prepared.
- composition of the present invention containing an expression vector containing a gene encoding the HAPLN1 protein
- a composition containing an expression vector containing the gene encoding the HAPLN1 protein was prepared using the jetPRIME Transfection Reagent Kit (114-15 product from PolyPlus). More specifically, a plasmid vector designed to express Human HAPLN1 was prepared using OriGene's RC209274 product (hereinafter referred to as “Human HAPLN1 ORF Clone”). It can express the genes it transports within the nucleus of the host cell.
- the jetPRIME Transfection Reagent Kit consists of jetPRIME buffer and jetPRIME Transfection reagent.
- the jetPRIME buffer is used to dilute the plasmid vector to be transported to host cells, and the jetPRIME Transfection reagent captures the plasmid vector in the form of a vesicle (liposome) and transmits it to the host cell. It works to penetrate into the inside.
- the Human HAPLN1 ORF Clone was diluted in jetPRIME buffer to prepare a composition of the present invention containing an expression vector containing the gene encoding the HAPLN1 protein.
- jetPRIME Transfection reagent is added and left for 10 minutes to capture the plasmid vector in the form of a vesicle (liposome) and then dropped onto the cells in culture. After culturing the cells for more than 48 hours, the increase in human HAPLN1 gene expression was confirmed by real-time qPCR, and the increase in human HAPLN1 protein expression was confirmed by Western blot.
- NHDF Normal Human Dermal Fibroblast
- Human dermal fibroblasts were cultured for 6 passages according to the manufacturer's protocol, then dispensed into 60 ⁇ dishes ( 5 It was incubated for 24 hours in an incubator under 37°C and 5% CO 2 conditions.
- the group was divided into a normal group without any treatment and a group treated with IL-1 ⁇ (10 ng/ml) for 24 hours to induce excessive production of ROS, and the composition of the present invention (rhHAPLN1) was administered to each group at a concentration of 0 and 2.5, respectively. , 5, and 10 ng/ml and then incubated for 24 hours.
- NRF2 an antioxidant marker
- NHLF Normal Human Lung Fibroblast
- the composition of the present invention (rhHAPLN1) was administered to the group treated with IL-1 ⁇ (10 ng/ml) for 24 hours to induce excessive production of ROS and the group not treated with IL-1 ⁇ (10 ng/ml). They were added at 0 and 5 ng/ml, respectively, and incubated for 24 hours.
- DCF-DA (20mM) was diluted at a ratio of 1:20,000, treated with each cell sample, and incubated for 10 minutes.
- the relative fluorescence level of reactive oxygen species (ROS) was measured for each sample using a Multi-Mode Reader (492/530nm, Synergy
- Human lung fibroblasts were cultured for 6 passages according to the manufacturer's protocol, then dispensed into 100 ⁇ dishes (6.5 It was incubated for 24 hours in an incubator under 37°C and 5% CO 2 conditions.
- the group was divided into a group treated with IL-1 ⁇ (10 ng/ml) to induce excessive production of ROS and a normal group without any treatment, and the composition of the present invention (rhHAPLN1) was administered to each group at 0 and 5 ng/ml, respectively. After treatment, it was incubated for 24 hours. Afterwards, cells were lysed with RIPA buffer (Radioimmunoprecipitation assay buffer) and protease/phosphatase inhibitor cocktail, and the total protein extract was separated by SDS-PAGE on a 10% acrylamide gel.
- RIPA buffer Radioimmunoprecipitation assay buffer
- protease/phosphatase inhibitor cocktail protease/phosphatase inhibitor cocktail
- cell extract (cell lysate) was loaded for immunoblot, and primary antibody against NRF2 (5% BSA-TBST (Bovine Serum Albumin-Tris-Buffered Saline & Polysorbate 20) was used for immunodetection. ) at a 1:1,000 dilution) and shaken overnight at 4°C. Incubate for 1 hour at room temperature with HRP (Horseradish Peroxidase)-conjugated secondary antibody (diluted 1:10,000 in 5% skim milk-TBST), and develop the protein band with ECL (Enhanced Chemiluminenscent, Femto) solution and then FUSION FX. (Vilber).
- HRP Hexeradish Peroxidase
- ECL Enhanced Chemiluminenscent, Femto
- HCEC Human Corneal Epithelial Cell
- HCEC Human Corneal Epithelial Cell
- 450mOsM medium was prepared and treated with 5M NaCl, and incubated for 48 hours.
- 314 mOsM is considered a normal control, but the relatively high osmotic pressure of 450 mOsM is a condition that induces the generation of active oxygen enough to be set as a dry eye syndrome model.
- 450mOsM medium which is a condition for inducing active oxygen generation.
- the composition of the present invention rhHAPLN1
- rhHAPLN1 phosphate buffered saline
- HCEC Human corneal epithelial cells
- the composition of the present invention (rhHAPLN1) (0, 10, 100 ng/ml) was added to the medium and incubated for 24 hours. Afterwards, the cells were lysed with RIPA buffer and protease/phosphatase inhibitor cocktail, the total protein extract was extracted, and proteins were loaded at 20 ⁇ g per well to confirm the expression of NRF2, GPx1, SOD1, and GAPDH, and 4-15% gradient acrylamide. Separated by SDS-PAGE on gel. These samples were shaken overnight at 4°C with primary antibody (1:1,000 dilution in 1% BSA-TBST) for immunodetection.
- the composition of the present invention (rhHAPLN1) was applied to normal human corneal epithelial cells under normal conditions (314 mOsM) and conditions that induced excessive production of reactive oxygen species (ROS) (450 mOsM).
- ROS reactive oxygen species
- NRF2, GPx1, and SOD1 proteins which are indicators of antioxidant activity, were comparable to cells under normal conditions in proportion to the content of the composition of the present invention (rhHAPLN1). Judging by the degree of expression, it can be seen that the composition of the present invention has the effect of significantly reducing ROS excessively generated under high osmotic pressure conditions.
- HMVEC-L Human Lung Microvascular Endothelial Cell
- EGM TM -2 complete medium 37°C and 5% CO 2 for 24 hours. Incubated for an hour.
- the groups were divided into a group treated with 30 ng/ml of the composition of the present invention (rhHAPLN1) for 2 hours and a group not treated.
- the cells were incubated with 20 ⁇ M DCF-DA for 20 minutes.
- H 2 O 2 was added as an external ROS at 250 ⁇ M to each sample. Fluorescence intensity was detected with a fluorescence photometer (Synergy HTX Multi-Mode Reader, BioTek Instruments) with an excitation wavelength of 485 nm and an emission wavelength of 535 nm.
- HMVEC-L Human lung microvascular endothelial cells
- EGM TM -2 complete medium 37°C and 5% CO 2 for 24 hours.
- the groups were divided into a group treated with 30 ng/ml of the composition of the present invention (rhHAPLN1) for 2 hours and a group not treated.
- H 2 O 2 was added as external ROS at 250 ⁇ M and incubated for 15 minutes ( Figure 8a) and 2 hours ( Figure 8b).
- the expression level of protein markers pAkt, NRF2, pVEGFR2 (Y1175), and VEGFR2, which are indicators of response to ROS, and GAPDH, an expression verification marker were detected by Western blot.
- Akt Akt
- NRF2 Akt
- the expression levels of pAkt and NRF2 may reflect the levels of ROS entering the cell. That is, when external ROS enters the cell, it activates NRF2 through PI3K/Akt.
- NRF2 When NRF2 is activated, it enters the cell nucleus as a transcription factor and transcribes various anti-oxidation enzymes.
- the expression of antioxidant enzymes is a process that takes several hours, and ultimately causes ROS entering the cells to induce oxidation of the cells.
- the human body naturally activates the antioxidant-related transcription factor (NRF2) through phosphorylation of Akt to remove ROS when it infiltrates (middle lane in Figure 8a).
- NRF2 antioxidant-related transcription factor
- the expression levels of pAkt and NRF2 did not increase significantly after 15 minutes of H 2 O 2 treatment compared to the case where the composition of the present invention was not pretreated ( right lane in Figure 8A).
- the fact that the expression levels of pAkt and NRF2 did not increase when pretreated with the composition of the present invention means that the amount of ROS entering the cells was small. This indicates that the composition of the present invention is much more effective than the natural state in blocking the entry of external ROS into cells. Of course, this will also lower the degree to which oxidation of cells is induced.
- VEGFR2 is a marker of endothelial cell proliferation, survival, and angiogenesis, and its decomposition begins when tyrosine, amino acid 1175, is phosphorylated.
- the composition of the present invention (rhHAPLN1) inhibits this phosphorylation and increases the amount of VEGFR2 displayed on the cell surface, thereby triggering signals for proliferation, survival, and angiogenesis in endothelial cells within blood vessels. That is, according to Figure 8b, after 2 hours of treatment with H 2 O 2 as ROS, the expression level of VEGFR2 is significantly lower than in the natural state without ROS, but when pretreated with the composition of the present invention (rhHAPLN1), the expression level is significantly recovered. It appears that
- composition of the present invention Confirmation of the ability of the composition of the present invention to prevent inhibition of cell proliferation by active oxygen in human lung microvascular endothelial cells against external ROS.
- HMVEC-L Human lung microvascular endothelial cells
- EGM TM -2 complete medium at 37°C and 5% CO 2 for 24 hours.
- the composition of the present invention (rhHAPLN1) was treated at constant concentrations (3, 10, 30 ng/ml) for 2 hours.
- H 2 O 2 was added to the cell samples at 1mM and incubated for 24 hours (A in FIG. 9) or 48 hours (B in FIG. 9).
- Cell proliferation was assessed using Cell Counting kit-8 (Dojindo).
- Figure 9 shows the change in cell proliferation rate by the composition of the present invention (rhHAPLN1) according to the dosage of the composition of the present invention when reactive oxygen species (ROS) are introduced directly from the outside of the cell. That is, the composition of the present invention (rhHAPLN1) was administered to human lung microvascular endothelial cells at 0, 3, 10, and 30. It can be seen that when ng/ml is added, the proliferation rate of cells is significantly increased compared to the case where the composition of the present invention is not treated. In particular, when the composition of the present invention was not treated after 48 hours, the cell proliferation rate was reduced to half compared to after 24 hours, but the composition of the present invention was used for 3 hours. When added at ng/ml or more, it appears to be maintained at about twice that level. This strongly suggests that the composition of the present invention can effectively prevent the action of ROS introduced from the outside to inhibit cell proliferation.
- ROS reactive oxygen species
- composition and method of the present invention by preventing and inhibiting the oxidation action of cells, the occurrence or progression of various diseases caused by excessive oxidation action of cells is fundamentally suppressed, thereby making it possible to prevent or treat related diseases.
- the reagent composition of the present invention can be used in research on various diseases related to oxidation and can help develop compositions for preventing or treating related diseases.
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Abstract
Disclosed herein are an antioxidant composition for cells, comprising hyaluronan and proteoglycan link protein 1 (HAPLN1) or the gene encoding same as an active ingredient, and a method for antioxidation in cells using the composition. According to the preset invention, cellular oxidation is prevented and suppressed to fundamentally inhibit the onset or progression of various diseases caused by excessive cellular oxidation, whereby the present invention offers the possibility of preventing or treating the diseases.
Description
본 발명은 히알루론산과 프로테오글리칸 연결 단백질 1(hyaluronan and proteoglycan link protein 1; HAPLN1) 또는 이를 코딩하는 유전자를 유효성분으로 포함하는 세포의 항산화 조성물 및 상기 조성물을 이용한 세포의 항산화 방법을 개시한다. 본 발명에 의하면 세포의 산화 작용을 예방 및 억제함으로써 세포의 산화 작용이 개입하여 유발하는 각종 질환의 발생 또는 진행을 근본적으로 억제하게 되어 해당 질환을 예방 내지 치료할 수 있는 가능성을 제시한다.The present invention discloses an antioxidant composition for cells containing hyaluronic acid and proteoglycan link protein 1 (HAPLN1) or a gene encoding the same as an active ingredient, and a method for antioxidantizing cells using the composition. According to the present invention, by preventing and suppressing the oxidation function of cells, the occurrence or progression of various diseases caused by intervention of the oxidation function of cells is fundamentally suppressed, suggesting the possibility of preventing or treating the diseases.
"활성 산소(Reactive Oxygen Species, ROS, '활성 산소종'으로 칭하는 경우도 있음)"는 산소(O2) 분자가 전자 1개를 받아 환원되면 안정적인 형태인 물(H2O) 분자로 전환될 때까지의 과정에서 생성되는 높은 에너지의 불안정한 산소들을 의미한다. 초과산화물 라디칼(Superoxide Anion Radical, ·O2-), 과산화수소(H2O2), 수산화 라디칼(Hydroxy Radical, HO·) 등이 대표적인 활성 산소로, 생체유지에 필수적인 호흡, 염증 등으로 인한 내부 요인과 자외선, 방사선, 흡연, 환경오염, 스트레스, 과식 등의 외부 요인으로 인해 발생된다.“Reactive Oxygen Species (ROS,” sometimes referred to as ‘reactive oxygen species’) is a chemical that can be converted into a stable water (H 2 O) molecule when an oxygen (O 2 ) molecule receives one electron and is reduced. This refers to high-energy, unstable oxygen generated during the process. Superoxide radicals (O 2- ), hydrogen peroxide (H 2 O 2 ), and hydroxy radicals (HO·) are representative active oxygen radicals, which are internal factors caused by breathing and inflammation, which are essential for maintaining the living body. It is caused by external factors such as ultraviolet rays, radiation, smoking, environmental pollution, stress, and overeating.
1950년대부터 Dr. Denham Harman, Dr. Earl Reece Stadtman 등에 의해 이러한 활성 산소들이 암, 고혈압, 당뇨, 동맥경화, 알츠하이머, 뇌졸중 등의 수많은 질병과 연관성이 있다고 보고되었는데, 이는 활성 산소들이 인체의 다른 분자들로부터 전자를 빼앗아 산화하여 안정적인 형태로 변하려는 성질로 인해 세포나 조직, DNA에 손상을 일으키기 때문이다.Since the 1950s, Dr. Denham Harman, Dr. It was reported by Earl Reece Stadtman and others that these free radicals are associated with numerous diseases such as cancer, high blood pressure, diabetes, arteriosclerosis, Alzheimer's, and stroke. This is because free radicals steal electrons from other molecules in the body and oxidize them into a stable form. This is because it causes damage to cells, tissues, and DNA due to its changing nature.
최근, 정상적으로 생성되거나 유지되는 낮은 농도의 활성 산소는 인체 면역체계 강화, 근육 재생, 당뇨 억제, 퇴행성 관절염 완화 등의 기능을 수행하는 것으로 알려져 세포 신호 전달과 항상성 유지에 반드시 필요한 물질로 인식되기도 하였다. 그러나, 비정상적으로 과도하게 생성되는 높은 농도의 활성 산소는 여전히 염증 발생, 변형 단백질 축적, 세포 이상증식으로 인한 종양 발생 등을 일으키므로 세포 내 활성 산소의 농도 조절에 관한 메커니즘이나 그에 대한 대책은 다양한 분야의 연구자들로부터 큰 관심을 받고 있다.Recently, low concentrations of reactive oxygen species, which are normally generated or maintained, are known to perform functions such as strengthening the human immune system, muscle regeneration, suppressing diabetes, and alleviating degenerative arthritis, and have been recognized as essential substances for cell signal transmission and homeostasis maintenance. However, the abnormally excessively generated high concentration of active oxygen still causes inflammation, accumulation of modified proteins, and tumor development due to abnormal cell proliferation, so the mechanism for controlling the concentration of active oxygen within cells and countermeasures are unknown in various fields. is receiving great attention from researchers.
인체는 이러한 활성 산소들을 제거하여 세포를 보호하기 위하여 초과산화물 불균등화효소(Superoxide Dismutase, SOD), 과산화수소 분해효소(Catalase), 글루타티온 과산화효소, 글루타티온 환원효소 등의 효소를 이용한 항산화 시스템을 원래 갖추고 있다. 그렇지만 이로는 충분하지 않으므로 사람들은 추가적으로 비타민류, 폴리페놀류, 미네랄류 등을 섭취하여 보완한다. 다만, 이들의 과다복용은 구역, 설사, 변비, 발진, 피로 등의 가벼운 부작용부터 뇌출혈, 폐암, 전립선암 등의 발생 증가와 같은 심각한 부작용을 초래하는 것으로 보고되어 있어서 종래부터 특정 질환을 앓고 있는 기저질환자의 복용은 논란이 되고 있다.The human body is originally equipped with an antioxidant system that uses enzymes such as superoxide dismutase (SOD), hydrogen peroxide decomposition enzyme (Catalase), glutathione peroxidase, and glutathione reductase to remove these active oxygen radicals and protect cells. . However, since this is not enough, people supplement by taking additional vitamins, polyphenols, and minerals. However, their overdose has been reported to cause mild side effects such as nausea, diarrhea, constipation, rash, and fatigue, as well as serious side effects such as increased incidence of cerebral hemorrhage, lung cancer, and prostate cancer, and thus, the Its use by people with diseases is controversial.
따라서, 비정상적으로 과다 생성된 활성 산소에 대해 직접적인 소거능을 갖춤은 물론, 부작용없이 인체의 항산화 체계를 강화시켜줄 수 있는 물질의 개발은 활성 산소로부터 유래되는 수많은 질병의 예방과 치료를 통해 인류보건에 크게 기여할 것으로 기대된다.Therefore, the development of a substance that not only has a direct scavenging ability against abnormally excessively generated free radicals, but can also strengthen the body's antioxidant system without side effects will greatly contribute to human health through the prevention and treatment of numerous diseases caused by free radicals. It is expected that it will contribute.
본 발명자들은 히알루론산과 프로테오글리칸 연결 단백질 1(hyaluronan and proteoglycan link protein 1; HAPLN1) 또는 이를 코딩하는 유전자에 관하여 깊이 연구한 결과 이들을 유효성분으로서 포함하는 조성물을 다양한 세포에 대해 처리할 때 세포의 비정상적인 산화작용을 미리 방지하거나 억제하여 정상화시킬 수 있다는 놀라운 기능들을 발견하여 본 발명을 완성하기에 이르렀다.The present inventors conducted an in-depth study on hyaluronic acid and proteoglycan link protein 1 (HAPLN1) or the gene encoding it, and found that when various cells were treated with a composition containing them as active ingredients, abnormal oxidation of cells occurred. The present invention was completed by discovering surprising functions that can prevent or inhibit the action and normalize it.
HAPLN1과 관련하여 예를 들어 특허문헌 1에는 골수간질세포(MSC, Marrow Stromal Cells)에 의해 분비되는 광범위한 개별 인자 중 하나로 HAPLN1 폴리펩티드를 개시하면서 이것을 염증성 질환 개체에 투여시 그 질환의 특징을 약화시킬 수 있음을 시사하고 있다. 그러나 세포의 항산화 작용에 관해서는 전혀 언급된 바가 없다.Regarding HAPLN1, for example, Patent Document 1 discloses HAPLN1 polypeptide as one of a wide range of individual factors secreted by bone marrow stromal cells (MSC, Marrow Stromal Cells), and when administered to an inflammatory disease subject, it can weaken the characteristics of the disease. It suggests that there is. However, nothing has been said about the antioxidant activity of cells.
또한 비특허문헌 1에는 "연골형성장애증 및 연골 link 단백질 결핍의 출생전후의 치사효과의 유전적 구제(Genetic Rescue of Chondrodysplasia and the Perinatal Lethal Effect of Cartilage Link Protein Deficiency)"에 관한 논문으로서 유전공학적으로 연골 link protein(Crtl1) 유전자를 파괴한 마우스에서는 연골형성장애가 나타나고 출생전후에 마우스가 사망하게 되나 Crtl1 null 마우스에 연골 특이적 link protein을 도입하여 과발현하면 비정상적인 연골 형성이 방지되고 출산 전후에 사망률이 낮아진다는 것과, Crtl1 전사체 및 그 단백질을 태아로부터 성체에 이르기까지 모든 기관에서 찾았다는 사실 등을 개시하고 있다. 그러나 여기에서도 세포에 대한 항산화 작용에 관해서는 전혀 언급된 바가 없다.In addition, Non-Patent Document 1 is a paper on "Genetic Rescue of Chondrodysplasia and the Perinatal Lethal Effect of Cartilage Link Protein Deficiency", which uses genetic engineering. Mice in which the cartilage link protein (Crtl1) gene is destroyed show cartilage dysplasia and the mice die before and after birth. However, if a cartilage-specific link protein is introduced and overexpressed in Crtl1 null mice, abnormal cartilage formation is prevented and the mortality rate before and after birth is reduced. and the fact that Crtl1 transcript and its protein were found in all organs from fetuses to adults. However, even here, nothing is mentioned about the antioxidant effect on cells.
생체 내의 세포에서 비정상적으로 과도하게 발생하는 산화 작용을 직접적으로 예방 내지 억제할 수 있다면 산화 작용 자체가 발병 원인으로 작용하거나 해당 산화작용이 이미 발병된 질환의 진행을 촉진하는 각종 질환에 대해 유효한 대비책이 될 것이며 이에 관한 요구는 항상 존재하고 있는 실정이다.If it is possible to directly prevent or suppress oxidation that occurs abnormally and excessively in cells within the body, it will be an effective countermeasure against various diseases in which the oxidation itself acts as a cause or promotes the progression of diseases that have already developed. This will happen, and the demand for this will always exist.
[선행기술문헌][Prior art literature]
[특허문헌][Patent Document]
(특허문헌 1) 특허문헌 1: 미국 공개특허 US 2013/0052198 (Patent Document 1) Patent Document 1: US Published Patent US 2013/0052198
[비특허문헌][Non-patent literature]
(비특허문헌 1)비특허문헌 1: Journal of Biological Chemistry.2003.Vol.278, No40.pp39214-29223(Non-patent Document 1)Non-patent Document 1: Journal of Biological Chemistry.2003.Vol.278, No40.pp39214-29223
본 발명은 히알루론산과 프로테오글리칸 연결 단백질 1(hyaluronan and proteoglycan link protein 1; HAPLN1) 또는 이를 코딩하는 유전자를 유효성분으로 포함하는 항산화 조성물 및 상기 조성물을 이용한 세포의 항산화 방법을 제공함으로써 세포의 산화 작용을 예방 또는 억제하여 세포의 산화 작용이 개입하여 유발되는 각종 질환의 발생 또는 진행을 근본적으로 억제하게 되어 해당 질환을 예방 내지 치료할 수 있는 가능성을 제시한다.The present invention provides an antioxidant composition containing hyaluronic acid and proteoglycan link protein 1 (HAPLN1) or a gene encoding the same as an active ingredient, and a method for antioxidantizing cells using the composition, thereby inhibiting the oxidation of cells. By preventing or inhibiting the occurrence or progression of various diseases caused by the intervention of cellular oxidation, it fundamentally suppresses the development or progression of the diseases, suggesting the possibility of preventing or treating the diseases.
또한 히알루론산과 프로테오글리칸 연결 단백질 1(hyaluronan and proteoglycan link protein 1; HAPLN1) 또는 이를 코딩하는 유전자를 유효성분으로 포함하는 시약 조성물을 제공함으로써 상기 질환 연구시에 사용되어 해당 질환의 메커니즘을 규명하고 관련 질환의 예방 또는 치료 조성물을 개발하는 데에 이바지하고자 한다.In addition, by providing a reagent composition containing hyaluronic acid and proteoglycan link protein 1 (HAPLN1) or the gene encoding it as an active ingredient, it can be used in research on the disease to identify the mechanism of the disease and treat related diseases. The aim is to contribute to the development of compositions for the prevention or treatment of.
상기 과제를 해결하기 위하여 본 발명은 다음과 같은 발명의 양상을 제공한다.In order to solve the above problems, the present invention provides the following aspects of the invention.
[1] 본 발명의 일 양상은 히알루론산과 프로테오글리칸 연결 단백질 1(hyaluronan and proteoglycan link protein 1; HAPLN1) 또는 이를 코딩하는 유전자를 유효성분으로 포함하는, 항산화 조성물에 관한 것이다.[1] One aspect of the present invention relates to an antioxidant composition comprising hyaluronic acid and proteoglycan link protein 1 (HAPLN1) or a gene encoding the same as an active ingredient.
[2] 또한 본 발명의 일 양상은 상기 조성물의 단백질이 서열번호 1의 아미노산 서열에 대해 80% 이상의 서열동일성을 갖는 것인 항산화 조성물에 관한 것이다.[2] Another aspect of the present invention relates to an antioxidant composition in which the protein of the composition has more than 80% sequence identity to the amino acid sequence of SEQ ID NO: 1.
[3] 또한 본 발명의 일 양상은 상기 조성물 중의 상기 유전자에 관한 핵산이 발현 벡터 내에 포함되어 있는 것인 항산화 조성물에 관한 것이다.[3] Another aspect of the present invention relates to an antioxidant composition in which the nucleic acid related to the gene in the composition is contained in an expression vector.
[4] 또한 본 발명의 일 양상은 상기 조성물이 세포의 산화작용을 예방 또는 억제하는 것인 항산화 조성물에 관한 것이다.[4] Another aspect of the present invention relates to an antioxidant composition in which the composition prevents or inhibits oxidation of cells.
[5] 또한 본 발명의 일 양상은 상기 조성물이 세포에서의 활성 산소의 과도한 발생을 억제하거나 이미 발생된 활성 산소를 감소시키는 것인 항산화 조성물에 관한 것이다.[5] Another aspect of the present invention relates to an antioxidant composition in which the composition suppresses excessive generation of active oxygen in cells or reduces active oxygen already generated.
[6] 또한 본 발명의 일 양상은 상기 조성물이 작용하는 세포가 섬유아세포, 상피세포 및 내피 세포로 이루어진 군에서 선택된 것인 항산화 조성물에 관한 것이다.[6] Another aspect of the present invention relates to an antioxidant composition wherein the cells on which the composition acts are selected from the group consisting of fibroblasts, epithelial cells, and endothelial cells.
[7] 또한 본 발명의 일 양상은 상기 조성물이 작용하는 세포가 in vitro 상태의 세포인 것인 항산화 조성물에 관한 것이다.[7] Additionally, one aspect of the present invention relates to an antioxidant composition in which the cells on which the composition acts are cells in an in vitro state.
[8] 또한 본 발명의 일 양상은 상기 조성물의 세포에 대한 1회 투여량이 0.1ng/ml 내지 1000ng/ml인 것인 조성물에 관한 것이다,[8] In addition, one aspect of the present invention relates to a composition in which a single dose of the composition to cells is 0.1 ng/ml to 1000 ng/ml,
[9] 또한 본 발명의 일 양상은 상기 조성물이 세포의 산화작용을 예방 또는 억제하기 위한 조성물의 주요 또는 보조 유효성분으로서 포함되는 것인 항산화 조성물에 관한 것이다.[9] Another aspect of the present invention relates to an antioxidant composition that is included as a main or auxiliary active ingredient in a composition for preventing or inhibiting oxidation of cells.
[10] 한편으로 본 발명의 또 다른 양상은 히알루론산과 프로테오글리칸 연결 단백질 1(hyaluronan and proteoglycan link protein 1; HAPLN1) 또는 이를 코딩하는 유전자를 유효성분으로 포함하는 조성물을 세포에 처리함으로써 세포의 산화작용을 예방 또는 억제하는 방법에 관한 것이다.[10] On the other hand, another aspect of the present invention is to treat cells with a composition containing hyaluronic acid and proteoglycan link protein 1 (HAPLN1) or the gene encoding it as an active ingredient, thereby promoting cell oxidation. It relates to methods of preventing or suppressing.
[11] 나아가 본 발명의 추가적인 양상은 항목 [1] 내지 항목 [9] 중 어느 한 항목에 따른 조성물 및 항목 [10]의 처리 지침을 포함하는 세포의 산화작용 예방 또는 억제용 키트에 관한 것이다.[11] Furthermore, an additional aspect of the present invention relates to a kit for preventing or inhibiting oxidation of cells, comprising the composition according to any one of items [1] to [9] and the processing instructions of item [10].
[12] 나아가 본 발명의 추가적인 양상은 항목 [1] 내지 항목 [9] 중 어느 한 항목에 따른 조성물을 포함하는 세포의 산화작용 예방 또는 억제와 관련된 실험용 시약 조성물에 관한 것이다.[12] Furthermore, an additional aspect of the present invention relates to an experimental reagent composition related to preventing or inhibiting oxidation of cells, comprising the composition according to any one of items [1] to [9].
상기한 바와 같이 본 발명의 조성물 및 방법에 의하면 세포의 산화 작용을 예방 및 억제함으로써 세포의 과도한 산화 작용이 개입하여 유발하는 각종 질환의 발생 또는 진행을 근본적으로 억제하게 되어 해당 질환을 예방 내지 치료할 수 있는 가능성을 획기적으로 높여준다.As described above, according to the composition and method of the present invention, the occurrence or progression of various diseases caused by excessive oxidation of cells is fundamentally suppressed by preventing and inhibiting the oxidation of cells, thereby preventing or treating the diseases. It dramatically increases the possibility.
또한 본 발명의 시약 조성물은 산화작용과 관련된 각종 질환 연구시에 사용되어 해당 질환의 메커니즘을 규명하고 해당 질환의 예방 또는 치료 조성물을 개발하는 데에 도움을 줄 수 있다.In addition, the reagent composition of the present invention can be used in research on various diseases related to oxidation to help identify the mechanism of the disease and develop a composition for preventing or treating the disease.
도 1은 정상 인간 피부 섬유아세포에 대해 IL-1β(10 ng/ml)를 처리하여 2시간 동안 활성 산소(ROS)의 과다생성을 유도한 조건에서 본 발명의 조성물(rhHAPLN1)을 각각 0, 2.5, 5, 10 ng/ml로 처리한 후 24시간 인큐베이트한 시점에서의 활성 산소의 발생을 억제하는 능력을 나타내는 실험 결과이다.Figure 1 shows normal human skin fibroblasts treated with IL-1β (10 ng/ml) to induce excessive production of reactive oxygen species (ROS) for 2 hours, and the composition of the present invention (rhHAPLN1) was administered at a concentration of 0 and 2.5, respectively. This is an experimental result showing the ability to suppress the generation of active oxygen when treated with , 5, and 10 ng/ml and incubated for 24 hours.
A: FACS(BD FACSLyricTM, BD Biosciences)를 이용하여 세포에서 ROS에 의해 생성된 형광 분포를 측정하여 ROS 수준의 변화를 나타낸 그래프이다. B: 본 발명의 조성물의 각각의 함량에 대한 DCF-DA 상대 강도를 배수로서 나타낸 그래프이다. C: 3회 실험을 수행하여 샘플당 ROS 수준에 대한 평균값을 정리하여 나타낸 표이다.A: This is a graph showing changes in ROS levels by measuring the distribution of fluorescence generated by ROS in cells using FACS (BD FACSLyric ™ , BD Biosciences). B: This is a graph showing the relative intensity of DCF-DA for each content of the composition of the present invention as a multiple. C: This is a table showing the average values of ROS levels per sample after performing three experiments.
도 2는 정상 인간 피부 섬유아세포에 대해 정상 조건 및 활성 산소(ROS)의 과다생성을 유도하는 조건 각각에 있어서, 본 발명의 조성물(rhHAPLN1)을 각각 0, 2.5, 5, 10 ng/ml로 처리한 후 24시간 인큐베이트한 시점에서의 활성 산소 억제능에 대한 항산화 관련 전사인자의 분자수준 변화를 나타내는 실험 결과이다. A: Multi-Mode Reader(488/520nm, Synergy|HTX)로 활성 산소에 의한 DCF-DA의 상대적인 형광 강도를 본 발명의 조성물의 각각의 함량에 대해 나타낸 그래프이다. B: 본 발명의 조성물의 각각의 함량에 대해 세포가 발현한 항산화 관련 단백질인 Nuclear Factor erythroid-2-related Factor 2 (NRF2)의 발현량을 웨스턴 블롯(Western blot)으로 확인한 결과이다( GAPDH는 단백질 발현이 정상적으로 수행되었음을 입증하는 참조 단백질 밴드에 해당함).Figure 2 shows normal human skin fibroblasts treated with the composition of the present invention (rhHAPLN1) at 0, 2.5, 5, and 10 ng/ml under normal conditions and conditions that induce excessive production of reactive oxygen species (ROS), respectively. This is the result of an experiment showing changes at the molecular level of antioxidant-related transcription factors for the ability to suppress free radicals after incubation for 24 hours. A: This is a graph showing the relative fluorescence intensity of DCF-DA due to active oxygen for each content of the composition of the present invention using Multi-Mode Reader (488/520nm, Synergy|HTX). B: This is the result of confirming the expression level of Nuclear Factor erythroid-2-related Factor 2 (NRF2), an antioxidant-related protein expressed by cells, by Western blot for each content of the composition of the present invention (GAPDH is a protein corresponds to the reference protein band demonstrating that expression was performed normally).
도 3은 정상 인간 폐 섬유아세포에 대해 아무 처리도 하지 않은 경우, 본 발명의 조성물(rhHAPLN1)만을 5 ng/ml 처리한 경우, 본 발명의 조성물 없이 IL-1β(10 ng/ml)를 처리하여 24시간동안 활성 산소(ROS)의 과다생성을 유도한 경우, 및 IL-1β(10 ng/ml)를 처리하여 ROS의 과다생성을 유도한 다음 본 발명의 조성물(rhHAPLN1) 5 ng/ml을 처리한 후 24시간 인큐베이트한 시점에서의 활성 산소의 억제능을 활성 산소(ROS)의 상대적인 생성 형광 정도를 통해 나타내는 실험 결과이다.Figure 3 shows normal human lung fibroblasts treated with no treatment, with only 5 ng/ml of the composition of the present invention (rhHAPLN1), and with IL-1β (10 ng/ml) without the composition of the present invention. When excessive production of reactive oxygen species (ROS) is induced for 24 hours, and excessive production of ROS is induced by treatment with IL-1β (10 ng/ml) and then treated with 5 ng/ml of the composition of the present invention (rhHAPLN1) This is an experimental result showing the inhibitory ability of reactive oxygen species after incubation for 24 hours through the relative degree of fluorescence generated by reactive oxygen species (ROS).
도 4는 정상 조건("×"로 표기) 및 활성 산소(ROS)의 과다생성을 유도한 조건("○"로 표기) 각각에 있어서, 정상 인간 폐 섬유아세포에 대해 본 발명의 조성물(rhHAPLN1)을 각각 0, 5 ng/ml로 처리한 후 24시간 인큐베이트한 시점에서의 활성 산소 억제능에 대한 항산화 관련 전사인자의 분자수준 변화를 나타내는 실험 결과이다. 샘플 중 세포가 발현한 항산화 관련 단백질인 NRF2의 발현량을 SDS-PAGE 및 면역검출법으로 확인한 사진이다.Figure 4 shows the composition of the present invention (rhHAPLN1) on normal human lung fibroblasts under normal conditions (marked with "×") and conditions inducing excessive production of reactive oxygen species (ROS) (marked with "○"). This is the result of an experiment showing changes at the molecular level of antioxidant-related transcription factors for reactive oxygen species suppression ability after treatment with 0 and 5 ng/ml, respectively, and incubation for 24 hours. This is a photo showing the expression level of NRF2, an antioxidant-related protein expressed by cells in the sample, using SDS-PAGE and immunodetection.
도 5는 인간 각막 상피세포에 대해 아무 처리도 하지 않은 샘플(정상 삼투압 314 mOsM), 및 5M NaCl로 450 mOsM 미디엄을 제조하여 처리함으로써 활성 산소(ROS)의 발생을 유도한 샘플들에 대해 본 발명의 조성물(rhHAPLN1)을 각각 0, 5, 10, 20, 50, 100 ng/ml로 처리한 후 24시간 인큐베이트한 시점에서의 활성 산소의 억제능을 나타내는 실험 결과이다. Multi-Mode Reader(485/528nm, Synergy|HTX)로 활성 산소에 의한 DCF-DA의 상대적인 형광 강도를 본 발명의 조성물의 각각의 함량에 대해 나타낸 그래프이다.Figure 5 shows a sample of human corneal epithelial cells without any treatment (normal osmolality 314 mOsM), and samples in which generation of reactive oxygen species (ROS) was induced by preparing and treating 450 mOsM medium with 5M NaCl according to the present invention. This is an experimental result showing the ability to suppress active oxygen when the composition (rhHAPLN1) was treated with 0, 5, 10, 20, 50, and 100 ng/ml, respectively, and incubated for 24 hours. This is a graph showing the relative fluorescence intensity of DCF-DA due to active oxygen for each content of the composition of the present invention using Multi-Mode Reader (485/528nm, Synergy|HTX).
도 6은 정상 조건(314 mOsM) 및 활성 산소(ROS)의 과다생성을 유도한 조건(450 mOsM) 각각에 있어서, 정상 인간 각막 상피세포에 대해 본 발명의 조성물(rhHAPLN1) 0, 10, 100 ng/ml로 각각 처리 후 24시간 인큐베이트한 시점에서의 활성 산소 억제능에 대한 항산화 관련 전사인자의 분자수준 변화를 나타내는 실험 결과이다. 샘플 중 세포가 발현한 항산화 관련 단백질들인 NRF2(Nuclear Factor erythroid-2-related factor 2), GPx1(Glutathione peroxidase 1), 및 SOD1(Superoxide dismutase 1)의 발현량을 SDS-PAGE 및 면역검출법으로 확인한 사진이다.Figure 6 shows 0, 10, and 100 ng of the composition of the present invention (rhHAPLN1) to normal human corneal epithelial cells under normal conditions (314 mOsM) and conditions inducing excessive production of reactive oxygen species (ROS) (450 mOsM), respectively. This is an experimental result showing changes at the molecular level of antioxidant-related transcription factors in terms of reactive oxygen species suppression ability at the time of incubation for 24 hours after each treatment with /ml. Photo showing the expression levels of antioxidant-related proteins expressed by cells in the sample, such as NRF2 (Nuclear Factor erythroid-2-related factor 2), GPx1 (Glutathione peroxidase 1), and SOD1 (Superoxide dismutase 1), using SDS-PAGE and immunodetection methods. am.
도 7은 인간 폐 미세혈관 내피세포에 대해 세포 외부로부터 직접 활성 산소(ROS)가 유입되었을 때 본 발명의 조성물(rhHAPLN1)에 의한 ROS 차단 성능을 나타내는 실험결과이다. 본 발명의 조성물(rhHAPLN1)을 전혀 첨가하지 않은 샘플, 및 본 발명의 조성물(rhHAPLN1) 30 ng/ml을 첨가한 샘플에 대해 H2O2 (250μM)를 가하여 그 시간에 따라 H2O2가 유도한 활성산소의 세포 내 증가율을 형광 광도계로 측정한 결과를 나타낸다(대조군(Control)은 본 발명의 조성물은 물론 H2O2도 가하지 않은 샘플임).Figure 7 shows experimental results showing the ROS blocking performance of the composition of the present invention (rhHAPLN1) when reactive oxygen species (ROS) is directly introduced from the outside of the cell to human lung microvascular endothelial cells. H 2 O 2 (250 μM) was added to the sample to which no composition of the present invention (rhHAPLN1) was added and to the sample to which 30 ng/ml of the composition of the present invention (rhHAPLN1) was added, and H 2 O 2 increased over time. Shows the results of measuring the intracellular increase rate of induced active oxygen using a fluorescence photometer (Control is a sample to which neither the composition of the present invention nor H 2 O 2 was added).
도 8a는 세포 외부로부터 직접 활성 산소(ROS)가 유입되었을 때 본 발명의 조성물(rhHAPLN1)에 의한 ROS 차단시 항산화 단백질 관련 단백질들의 분자수준 변화를 나타내는 실험결과이다. 인간 폐 미세혈관 내피세포에 대해 본 발명의 조성물(rhHAPLN1)을 전혀 첨가하지 않은 샘플 및 본 발명의 조성물(rhHAPLN1) 30 ng/ml을 첨가한 샘플에 대해 H2O2 (250μM)를 가한 후 15분 경과에 따라 ROS에 대해 반응하는, 즉 항산화 작용의 지표가 되는 각종 단백질 마커(pAkt, NRF2, pVEGFR2(Y1175), 및 VEGFR2)의 발현량을 웨스턴 블롯으로 나타낸 사진이다.Figure 8a is an experimental result showing changes in the molecular level of antioxidant protein-related proteins when ROS is blocked by the composition of the present invention (rhHAPLN1) when reactive oxygen species (ROS) are introduced directly from the outside of the cell. 15 after adding H 2 O 2 (250 μM) to human lung microvascular endothelial cells to a sample to which no composition of the present invention (rhHAPLN1) was added and to a sample to which 30 ng/ml of the composition of the present invention (rhHAPLN1) was added. This is a photograph showing the expression level of various protein markers (pAkt, NRF2, pVEGFR2(Y1175), and VEGFR2) that respond to ROS over time, that is, indicators of antioxidant activity, using Western blot.
도 8b는 도 8a의 실험 조건에 있어서, 본 발명의 조성물(rhHAPLN1)을 전혀 첨가하지 않은 샘플 및 본 발명의 조성물(rhHAPLN1) 30 ng/ml을 첨가한 샘플에 대해 H2O2 (250μM)를 가한 후 2시간 경과 후의 ROS에 대해 반응하는, 즉 항산화 작용의 지표가 되는 단백질 마커 중 특히 VEGFR2의 발현량을 웨스턴 블롯으로 나타낸 사진이다.Figure 8b shows H 2 O 2 (250 μM) for the sample to which no composition of the present invention (rhHAPLN1) was added and to the sample to which 30 ng/ml of the composition of the present invention (rhHAPLN1) was added under the experimental conditions of Figure 8a. This is a Western blot photo showing the expression level of VEGFR2, a protein marker that responds to ROS 2 hours after application, that is, an indicator of antioxidant activity.
도 9는 세포 외부로부터 직접 활성 산소(ROS)가 유입되었을 때 본 발명 조성물의 투여량에 따른 본 발명의 조성물(rhHAPLN1)에 의한 세포 증식율의 변화를 나타내는 실험결과이다. 인간 폐 미세혈관 내피세포에 대해 본 발명의 조성물(rhHAPLN1)을 0, 3, 10, 및 30 ng/ml을 첨가한 샘플 각각에 대해 H2O2 (1mM)를 가하고 24시간 후(A) 및 48시간 후(B)에 있어서 세포의 증식률을 Cell Counting kit-8 (Dojindo)로 평가하고 그래프로 각각 나타내었다(대조군은 본 발명의 조성물은 물론 H2O2도 가하지 않은 샘플임).Figure 9 is an experimental result showing the change in cell proliferation rate by the composition of the present invention (rhHAPLN1) according to the dosage of the composition of the present invention when reactive oxygen species (ROS) are introduced directly from the outside of the cell. The composition of the present invention (rhHAPLN1) was administered to human lung microvascular endothelial cells at 0, 3, 10, and 30 For each sample to which ng/ml was added, H 2 O 2 (1mM) was added, and the proliferation rate of cells was evaluated using Cell Counting kit-8 (Dojindo) at 24 hours (A) and 48 hours (B), and the graph (The control group is a sample to which neither the composition of the present invention nor H 2 O 2 was added).
본 발명의 명세서에 있어서, "rhHAPLN1"는 recombinant human HAPLN1의 약자로서 재조합 인간 HAPLN1을 나타낸다.In the specification of the present invention, “rhHAPLN1” is an abbreviation for recombinant human HAPLN1 and represents recombinant human HAPLN1.
본 발명의 명세서에 있어서, "DCF-DA 어세이"는 DCF-DA(Dichlorofluorescein Diacetate)가 세포막을 통해 확산하여 세포 내 에스테라제(Esterase)에 의해 가수분해됨으로써 비 형광성 DCF-H가 되며, 세포 내에 ROS가 존재할 경우 매우 빠르게 높은 형광을 띄는 DCF로 산화되는 원리를 가진 물질인 것을 이용하여 ROS의 생성을 확인하기 위해 널리 쓰이는 분석법이다.In the specification of the present invention, "DCF-DA Assay" means that DCF-DA (Dichlorofluorescein Diacetate) diffuses through the cell membrane and is hydrolyzed by intracellular esterase to become non-fluorescent DCF-H, This is a widely used analysis method to confirm the generation of ROS, using the principle that when ROS is present, it is oxidized very quickly to highly fluorescent DCF.
본 발명의 명세서에 있어서, "DCF-H는 2,7-dichlorodihydrofluorescein의 약자이다.In the specification of the present invention, "DCF-H is an abbreviation for 2,7-dichlorodihydrofluorescein.
본 발명의 명세서에 있어서 "IL(인터루킨)-1β"는 pro-inflammatory cytokine의 일종으로, 염증반응 전사인자인 NFκB(Nuclear Factor kappa B)를 활성화시켜 비정상적인 염증반응을 매개함으로써, 각 세포들에서 ROS를 과다생성시키기 위해 처리하는 물질을 지칭한다.In the specification of the present invention, "IL (interleukin)-1β" is a type of pro-inflammatory cytokine, which mediates an abnormal inflammatory response by activating NFκB (Nuclear Factor kappa B), an inflammatory response transcription factor, thereby generating ROS in each cell. It refers to a substance that is processed to produce excessive amounts of .
본 발명의 명세서에 있어서, "NRF2"는 Nuclear Factor erythroid-2-related Factor 2의 약자로서 이는 항산화 관련 유전자들의 발현에 영향을 주는 대표적인 전사인자로, 간, 폐, 소화기관, 뇌, 피부 등 인체 주요 조직에 분포하는 단백질이다.In the specification of the present invention, "NRF2" is an abbreviation for Nuclear Factor erythroid-2-related Factor 2, which is a representative transcription factor that affects the expression of antioxidant-related genes, and is found in the human body, such as the liver, lungs, digestive organs, brain, and skin. It is a protein distributed in major tissues.
본 발명의 명세서에 있어서, "GPx1"는 Glutathione peroxidase 1의 약자로서, 이는 과산화수소를 물로 환원시켜주는 역할을 하는 가장 중요한 항산화 효소 중 하나인 인자이다.In the specification of the present invention, "GPx1" is an abbreviation for Glutathione peroxidase 1, which is one of the most important antioxidant enzymes that reduces hydrogen peroxide to water.
본 발명의 명세서에 있어서, "SOD1"는 Superoxide dismutase 1의 약자로서 이는 세포질에 존재하는 항산화 효소로서 과산화물 음이온(Superoxide anion)을 과산화수소로 전환하는 반응을 촉매한다.In the specification of the present invention, “SOD1” is an abbreviation for Superoxide dismutase 1, which is an antioxidant enzyme present in the cytoplasm that catalyzes the reaction of converting superoxide anion into hydrogen peroxide.
본 발명의 명세서에 있어서, "Akt"는 Protein Kinase B를 지칭하는 것으로서 serine threonine kinase의 일종이며, 합성된 PIP3의 pleckstrin-homology(PH)와 직접 접촉하게 되고 Thr308과 Ser473 부위에서 인산화가 일어나면서, 세포형질막에 결집하고 활성화되는데, 신호전달 조건에 따라 활성 산소의 생산을 억제하기도 촉진하기도 하는 인자이지만 ROS 과다시에 NRF2를 활성화시키는 작용을 주로 하는 것으로 파악된다. In the specification of the present invention, "Akt" refers to Protein Kinase B, a type of serine threonine kinase, and comes into direct contact with the pleckstrin-homology (PH) of synthesized PIP3, and phosphorylation occurs at the Thr308 and Ser473 sites, It binds to the cell plasma membrane and is activated. It is a factor that suppresses or promotes the production of reactive oxygen species depending on the signal transduction conditions, but it is understood that it mainly acts to activate NRF2 when ROS is excessive.
본 발명의 명세서에 있어서, "VEGFR2"는 Vascular Endothelial Growth Factor Receptor 2의 약자로서 이는 혈관내피세포에서 증식(Proliferation), 생존(Survival) 및 혈관생성(Angiogenesis)과 관련된 인자로서, 산화 스트레스에 의해 감소된다.In the specification of the present invention, "VEGFR2" is an abbreviation for Vascular Endothelial Growth Factor Receptor 2, which is a factor related to proliferation, survival, and angiogenesis in vascular endothelial cells, and is reduced by oxidative stress. do.
본 발명의 일 양상은 히알루론산과 프로테오글리칸 연결 단백질 1(hyaluronan and proteoglycan link protein 1; HAPLN1) 또는 이를 코딩하는 유전자를 유효성분으로 포함하는, 항산화 조성물에 관한 것이다.One aspect of the present invention relates to an antioxidant composition comprising hyaluronic acid and proteoglycan link protein 1 (HAPLN1) or a gene encoding the same as an active ingredient.
또한 본 발명의 일 구체예에 따르면 상기 본 발명의 조성물에 있어서 서열번호 1로 나타나는 인간 재조합 HAPLN1 단백질일 수 있다. 이와 관련하여 본 발명의 HAPLN1 단백질은 항산화 기능을 유지하는 한 서열번호 1의 아미노산 서열에 대해 80%, 바람직하게는 85%, 더 바람직하게는 90%, 더더욱 바람직하게는 95%, 가장 바람직하게는 100%의 서열동일성을 갖는 단백질일 수 있다. Additionally, according to one embodiment of the present invention, it may be the human recombinant HAPLN1 protein represented by SEQ ID NO: 1 in the composition of the present invention. In this regard, the HAPLN1 protein of the present invention is 80%, preferably 85%, more preferably 90%, even more preferably 95%, and most preferably 80% of the amino acid sequence of SEQ ID NO: 1 as long as it maintains the antioxidant function. It may be a protein with 100% sequence identity.
또한 본 발명의 일 구체예에 따르면 상기 본 발명의 조성물 중의 HAPLN1 단백질을 코딩하는 유전자에 관한 핵산은 발현 벡터 내에 포함되어 상기 조성물 중에 포함될 수 있다.Additionally, according to one embodiment of the present invention, the nucleic acid related to the gene encoding the HAPLN1 protein in the composition of the present invention may be included in the composition by being included in an expression vector.
또한 본 발명의 일 구체예에 따르면 상기 본 발명의 조성물은 다양한 세포의 산화작용을 예방 또는 억제하는 효과를 나타내며, 이와 같은 세포의 산화작용을 예방 또는 억제하는 효과는 세포에서의 활성 산소의 과도한 발생을 억제하거나 이미 발생된 활성 산소를 감소시키는 효과를 포함할 뿐만 아니라 외부에서 유입되는 활성 산소를 유입 전 및/또는 유입 후에 차단시키는 효과를 포함한다.In addition, according to one embodiment of the present invention, the composition of the present invention exhibits the effect of preventing or suppressing the oxidation action of various cells, and the effect of preventing or suppressing the oxidation action of such cells is caused by excessive generation of active oxygen in the cell. It not only includes the effect of suppressing or reducing active oxygen that has already been generated, but also includes the effect of blocking active oxygen flowing in from the outside before and/or after the influx.
또한 본 발명에 있어서, 본 발명의 조성물이 작용하는 상기 세포는 특별히 한정되지 않으나 바람직하게는 섬유아세포, 상피세포 및 내피세포 등이며 in vivo, in vitro, ex vivo, in situ 등 다양한 상황에서의 세포를 포함하나 바람직하게는 in vitro일 수 있다.In addition, in the present invention, the cells on which the composition of the present invention acts are not particularly limited, but are preferably fibroblasts, epithelial cells, and endothelial cells, and can be used in various situations such as in vivo, in vitro, ex vivo, and in situ. It includes, but is preferably in vitro.
또한 본 발명의 일 구체예에 따르면 상기 본 발명의 조성물은 세포에 대한 1회 투여량이 0.1ng/ml 내지 1000ng/ml, 바람직하게는 1ng/ml 내지 500ng/ml, 더 바람직하게는 2.0ng/ml 내지 100ng/ml, 더욱 더 바람직하게는 2.5ng/ml 내지 50ng/ml, 가장 바람직하게는 3.0ng/ml 내지 30ng/ml이다.In addition, according to one embodiment of the present invention, the composition of the present invention has a single dose to cells of 0.1 ng/ml to 1000 ng/ml, preferably 1 ng/ml to 500 ng/ml, more preferably 2.0 ng/ml. to 100 ng/ml, even more preferably 2.5 ng/ml to 50 ng/ml, and most preferably 3.0 ng/ml to 30 ng/ml.
한편으로 본 발명의 일 구체예에서는 상기 본 발명의 조성물이 세포의 산화작용을 예방 또는 억제하기 위한 조성물의 주요 또는 보조 유효성분으로서 포함될 수 있다.On the other hand, in one embodiment of the present invention, the composition of the present invention may be included as a main or auxiliary active ingredient in a composition for preventing or inhibiting oxidation of cells.
나아가 본 발명의 일 양상에 따르면 히알루론산과 프로테오글리칸 연결 단백질 1(hyaluronan and proteoglycan link protein 1; HAPLN1) 또는 이를 코딩하는 유전자를 유효성분으로 포함하는 조성물을 세포에 처리함으로써 세포의 산화작용을 예방 또는 억제하는 방법에 관한 것이다.Furthermore, according to one aspect of the present invention, preventing or inhibiting oxidation of cells by treating cells with a composition containing hyaluronic acid and proteoglycan link protein 1 (HAPLN1) or a gene encoding the same as an active ingredient. It's about how to do it.
또한 본 발명의 일 양상에 따르면 상기 본 발명의 조성물 및 처리 지침을 포함하는 세포의 산화작용 예방 또는 억제용 키트에 관한 것이다.In addition, according to one aspect of the present invention, it relates to a kit for preventing or inhibiting oxidation of cells, including the composition of the present invention and processing instructions.
또한 본 발명의 일 양상에 따르면 상기 본 발명의 조성물을 포함하는, 세포의 산화작용 예방 또는 억제와 관련된 실험용 시약 조성물에 관한 것이다.In addition, according to one aspect of the present invention, it relates to a reagent composition for experiments related to preventing or inhibiting oxidation of cells, comprising the composition of the present invention.
나아가 본 발명은 세포의 산화작용이 유발하는 질환의 예방 또는 치료용 약학적 조성물의 제조에 사용하기 위한 상기 본 발명의 조성물의 사용에 관한 것이다. Furthermore, the present invention relates to the use of the composition of the present invention for use in the production of a pharmaceutical composition for preventing or treating diseases caused by cellular oxidation.
특정 구체예에서, 상기 약학적 조성물은 일반적으로 분자 및 약제학적으로 허용되는 담체를 포함할 수 있다. 본 명세서에서 사용된 용어 "약제학적으로 허용되는 담체"는 약학적 투여와 양립 가능한 식염수, 용매, 분산 매질, 코팅, 항균 및 항진균제, 등장화제 및 흡수 지연제 등을 포함한다. 보충 활성 화합물도 또한 조성물에 포함될 수 있다. 다시 말하면 본 발명의 약학적 조성물은 약학적으로 허용가능한 담체, 희석제, 결합제, 붕해제, 활택제, 및 이들의 임의의 조합으로 이루어진 군에서 선택된 약학적 첨가제를 더 포함할 수 있다.In certain embodiments, the pharmaceutical composition may generally include a molecule and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” includes saline solutions, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc. compatible with pharmaceutical administration. Supplementary active compounds may also be included in the composition. In other words, the pharmaceutical composition of the present invention may further include pharmaceutical additives selected from the group consisting of pharmaceutically acceptable carriers, diluents, binders, disintegrants, lubricants, and any combinations thereof.
상기 약학적 조성물은 의도된 투여 경로와 양립할 수 있도록 제형화될 수 있다. 바람직하게는 본 발명의 조성물은 점안액, 연고, 정제, 알약, 캡슐, 트로키제, 흡입제, 주사제, 패치제, 좌약으로 이루어진 군에서 선택되는 제형일 수 있다.The pharmaceutical composition may be formulated to be compatible with the intended route of administration. Preferably, the composition of the present invention may be in a dosage form selected from the group consisting of eye drops, ointments, tablets, pills, capsules, troches, inhalants, injections, patches, and suppositories.
투여 경로의 예는 비경구, 예를 들어 정맥 내, 피내, 피하, 경구 (예를 들어, 흡입), 경피 (국소), 경점막 및 직장 투여를 포함한다. 바람직하게는 비경구 투여용도이다. Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Preferably it is for parenteral administration.
비경구, 피내 또는 피하 적용에 사용되는 용액 또는 현탁액에는 다음 성분이 포함될 수 있다: 주사용수, 식염수, 고정 오일, 폴리에틸렌 글리콜, 글리세린, 프로필렌 글리콜 또는 기타 합성 용매와 같은 멸균 희석제; 벤질 알코올 또는 메틸 파라벤과 같은 항균제; 아스코르브산 또는 중아황산나트륨과 같은 항산화제; 에틸렌디아민테트라아세트산과 같은 킬레이트제; 아세테이트, 시트레이트 또는 포스페이트와 같은 완충액 및 염화나트륨 또는 덱스트로스와 같은 긴장성 조절 제제. pH는 염산 또는 수산화 나트륨과 같은 산 또는 염기로 조정할 수 있다. 비경구 제제는 앰플, 일회용 주사기 또는 유리 또는 플라스틱으로 만든 다회 용량 바이알에 넣을 수 있다. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following ingredients: sterile diluents such as water for injection, saline, fixative oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; Antibacterial agents such as benzyl alcohol or methyl paraben; Antioxidants such as ascorbic acid or sodium bisulfite; Chelating agents such as ethylenediaminetetraacetic acid; Buffers such as acetate, citrate or phosphate and tonicity-adjusting agents such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be placed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.
주사용으로 적합한 상기 약학적 조성물은 멸균된 주사용 용액 또는 분산액의 즉석 제조(extemporaneous preparation)를 위한 멸균 수용액 (수용성인 경우) 또는 분산액 및 멸균 분말을 포함한다. 정맥 내 투여에 적합한 담체는 생리 식염수, 정균수, 또는 인산염 완충 식염수 (PBS)를 포함한다. 모든 경우에 조성물은 무균이어야하고 주사가 용이할 정도로 유동적이어야한다. 제조 및 보관 조건 하에서 안정적이어야 하며 박테리아 및 곰팡이와 같은 미생물의 오염 작용에 대해 보존되어야한다. 담체는 예를 들어 물, 에탄올, 폴리올 (예를 들어, 글리세롤, 프로필렌 글리콜, 및 액체 폴리에틸렌 글리콜 등) 및 이들의 적합한 혼합물을 함유하는 용매 또는 분산 매질일 수 있다. 예를 들어, 레시틴과 같은 코팅의 사용, 분산의 경우 필요한 입자 크기의 유지 및 계면 활성제의 사용에 의해 적절한 유동성이 유지될 수 있다. 미생물 작용의 예방은 예를 들어 파라벤, 클로로부탄올, 페놀, 아스코르브산, 티메로살 등과 같은 다양한 항균 및 항진균제에 의해 달성될 수 있다. 많은 경우에 등장제, 예를 들어 당, 만니톨과 같은 폴리알콜, 소르비톨, 염화나트륨을 조성물에 포함시키는 것이 바람직할 것이다. 주사 가능한 조성물의 장기간 흡수는 흡수를 지연시키는 제제, 예를 들어 알루미늄 모노스테아레이트 및 젤라틴을 조성물에 포함시킴으로써 도모할 수 있다. The pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Carriers suitable for intravenous administration include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to facilitate injection. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and molds. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, appropriate fluidity can be maintained by the use of a coating such as lecithin, maintenance of the required particle size in case of dispersion, and use of a surfactant. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases it will be desirable to include isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, in the composition. Prolonged absorption of injectable compositions can be facilitated by including in the composition agents that delay absorption, such as aluminum monostearate and gelatin.
멸균 주사용 용액은 필요에 따라 상기 열거된 성분의 하나 또는 조합과 함께 적절한 용매에 필요한 양의 활성 화합물을 혼입시킨 다음 여과 멸균함으로써 제조할 수 있다. 일반적으로 분산액은 활성 화합물을 기본 분산 매질과 위에서 열거한 다른 필요 성분을 포함하는 멸균 비히클에 통합하여 제조한다. 멸균 주사 용액의 제조를 위한 멸균 분말의 경우, 바람직한 제조 방법은 진공 건조 및 동결 건조로서, 활성 성분의 분말과 이전에 멸균 여과된 용액으로부터 원하는 임의의 추가 성분을 생성한다. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Dispersions are generally prepared by incorporating the active compound into a sterile vehicle containing the basic dispersion medium and the other required ingredients listed above. For sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze drying, which yield a powder of the active ingredient and any additional ingredients desired from a previously sterile filtered solution.
경구 조성물은 일반적으로 불활성 희석제 또는 식용 담체를 포함한다. 경구 치료 투여의 목적을 위해, 활성 화합물은 부형제와 혼입될 수 있고 정제, 트로키 또는 캡슐, 예를 들어 젤라틴 캡슐의 형태로 사용될 수 있다. 구강용 조성물은 또한 구강 세정제로 사용하기 위한 유체 담체를 사용하여 제조할 수 있다. 약제학적으로 적합한 결합제 및/또는 보조제 물질이 조성물의 일부로 포함될 수 있다. 정제, 알약, 캡슐, 트로키 등은 다음 성분 또는 유사한 성질의 화합물 중 임의의 것을 함유할 수 있다: 바인더, 예컨대 미세결정질 셀룰로스, 트라가칸트 검 또는 젤라틴; 전분 또는 락토스와 같은 부형제, 알긴산, 프리모겔 또는 옥수수 전분과 같은 붕해제; 마그네슘 스테아레이트 또는 스테로테스와 같은 윤활제; 콜로이드성 이산화 규소와 같은 활택제; 수크로스 또는 사카린과 같은 감미제; 또는 페퍼민트, 메틸 살리실레이트, 또는 오렌지 향료와 같은 향료. Oral compositions generally include an inert diluent or edible carrier. For the purpose of oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of tablets, troches or capsules, for example gelatin capsules. Oral compositions can also be prepared using fluid carriers for use as oral rinses. Pharmaceutically suitable binders and/or adjuvant substances may be included as part of the composition. Tablets, pills, capsules, troches, etc. may contain any of the following ingredients or compounds of similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; Excipients such as starch or lactose, disintegrants such as alginic acid, Primogel or corn starch; Lubricants such as magnesium stearate or steroids; Glidants such as colloidal silicon dioxide; Sweeteners such as sucrose or saccharin; Or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
흡입에 의한 투여를 위해 화합물은 적절한 추진제, 예를 들어 이산화탄소와 같은 가스, 또는 네불라이저를 함유하는 압력 용기 또는 디스펜서로부터 에어로졸 스프레이의 형태로 전달된다.For administration by inhalation, the compound is delivered in the form of an aerosol spray from a pressure vessel or dispenser containing a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.
전신 투여는 또한 경점막 또는 경피 수단에 의해 이루어질 수 있다. 경점막 또는 경피 투여의 경우, 침투할 장벽에 적합한 침투제가 제형에 사용된다. 이러한 침투제는 일반적으로 당해 기술분야에 공지되어 있으며, 예를 들어 경점막 투여용, 세제, 담즙 염, 및 푸시드산 유도체를 포함한다. 경점막 투여는 비강 스프레이 또는 좌약을 사용하여 수행할 수 있다. 경피 투여를 위해 활성 화합물은 당해 기술분야에 일반적으로 알려진 바와 같이 연고, 고약, 겔 또는 크림으로 제형화된다. Systemic administration can also be achieved by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrating agent suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished using a nasal spray or suppository. For transdermal administration the active compounds are formulated as ointments, salves, gels or creams, as is generally known in the art.
화합물은 또한 좌약 (예를 들어, 코코아 버터 및 기타 글리세리드와 같은 통상적 인 좌약베이스와 함께) 또는 직장 전달을 위한 체류 관장의 형태로 제조될 수 있다. The compounds may also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
세포 배양 분석 및 동물 연구에서 얻은 데이터는 인간에게 사용하기 위한 다양한 용량을 제형화하는 데 사용할 수 있다. 이러한 화합물의 투여량은 바람직하게는 독성이 거의 또는 전혀없는 ED50을 포함하는 순환 농도 범위 내에 있다. 투여량은 사용되는 투여 형태 및 사용되는 투여 경로에 따라 이 범위 내에서 달라질 수 있다. Data obtained from cell culture assays and animal studies can be used to formulate various doses for use in humans. The dosage of these compounds is preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity. Dosages may vary within this range depending on the dosage form used and the route of administration used.
본 발명의 조성물의 치료적 유효량 (즉, 유효 투여량)은 선택된 환자의 상황에 따라 달라진다. 예를 들어, 약 1pg 내지 1000mg 범위의 단일 용량이 투여될 수 있다; 일부 구체예에서, 10, 30, 100, 또는 1000 pg, 또는 10, 30, 100, 또는 1000 ng, 또는 10, 30, 100, 또는 1000 μg, 또는 10, 30, 100 또는 1000 mg이 투여될 수 있다. The therapeutically effective amount (i.e., effective dosage) of the composition of the present invention will vary depending on the circumstances of the selected patient. For example, a single dose ranging from about 1 pg to 1000 mg may be administered; In some embodiments, 10, 30, 100, or 1000 pg, or 10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 μg, or 10, 30, 100, or 1000 mg may be administered. there is.
일부 구체예에서, 1ng/ml 내지 100μg/ml, 바람직하게는 3ng/ml 내지 50μg/ml, 더 바람직하게는 5ng/ml 내지 500ng/ml, 특히 바람직하게는 10ng/ml 내지 200ng/ml의 약학적 조성물이 투여할 수 있다. 상기 약학적 조성물은 1 일 1 회 이상부터 1 주일에 1 회 이상 투여할 수 있으며 격일로 한 번씩을 포함한다. 숙련된 기술자는 개체를 효과적으로 치료하기 위해 필요한 용량 및 시기에 특정 요인이 영향을 미칠 수 있음을 인식할 것인데 이에는 질병 또는 장애의 중증도, 이전의 치료, 개체의 일반적인 건강 및/또는 연령 및 기타 기존 질환을 포함하지만 이에 제한되지 않는다. 더욱이, 본 발명의 분자의 치료적 유효량으로 개체를 치료하는 것은 단일 치료를 포함할 수 있거나, 바람직하게는 일련의 치료를 포함할 수 있다.In some embodiments, the pharmaceutical dose is between 1 ng/ml and 100 μg/ml, preferably between 3 ng/ml and 50 μg/ml, more preferably between 5 ng/ml and 500 ng/ml, particularly preferably between 10 ng/ml and 200 ng/ml. The composition can be administered. The pharmaceutical composition can be administered from once a day to once a week, including once every other day. The skilled technician will recognize that certain factors may affect the dosage and timing required to effectively treat an individual, including the severity of the disease or disorder, prior treatment, the individual's general health and/or age, and other pre-existing conditions. Including, but not limited to, disease. Moreover, treating an individual with a therapeutically effective amount of a molecule of the invention may include a single treatment or, preferably, may include a series of treatments.
본 발명의 조성물은 환자의 상황에 따라 그 투여량이 5 mg/kg/주 내지 500 mg/kg/주, 예를 들어 5 mg/kg/주, 10 mg/kg/주, 15 mg/kg/주, 20 mg/kg/주, 25 mg/kg/주, 30 mg/kg/주, 35 mg/kg/주, 40 mg/kg/주, 45 mg/kg/주, 50 mg/kg/주, 55mg/kg/주, 60mg/kg/주, 65mg/kg/주, 70mg/kg/주, 75mg/kg/주, 80mg/kg/주, 85mg/kg/주, 90mg/kg/주, 95mg/kg/주, 100mg/kg/주, 150mg/kg/주, 200mg/kg/주, 250mg/kg/주, 300mg/kg/주, 350mg/kg/주, 400mg/kg/주, 450mg/kg/주 및 500mg/kg/주의 범위 내에 있다. 특정 구체예에서, 본 발명에 따른 이중작용성 분자의 투여량은 10 mg/kg/주 내지 200 mg/kg/주, 20 mg/kg/주 내지 150 mg/kg/주 또는 25 mg/kg/주 내지 100 mg/kg/주의 범위 내에 있다. 특정 구체예에서, 본 발명의 조성물은 2 주 내지 6 개월, 예를 들어 2 주, 3 주, 4 주, 5 주, 6 주, 7 주, 8 주, 9 주, 10주, 11 주, 12 주, 13 주, 26 주, 6 개월, 8 개월, 10 개월 또는 1 년 이상 동안 주당 1x 회 투여된다. 특정 구체예에서, 본 발명의 조성물은 주당 2x 회 투여된다. 다른 구체예에서, 본 발명의 조성물은 격주로 투여된다.The composition of the present invention has a dosage of 5 mg/kg/week to 500 mg/kg/week, for example, 5 mg/kg/week, 10 mg/kg/week, and 15 mg/kg/week, depending on the patient's situation. , 20 mg/kg/week, 25 mg/kg/week, 30 mg/kg/week, 35 mg/kg/week, 40 mg/kg/week, 45 mg/kg/week, 50 mg/kg/week, 55mg/kg/week, 60mg/kg/week, 65mg/kg/week, 70mg/kg/week, 75mg/kg/week, 80mg/kg/week, 85mg/kg/week, 90mg/kg/week, 95mg/ kg/wk, 100mg/kg/wk, 150mg/kg/wk, 200mg/kg/wk, 250mg/kg/wk, 300mg/kg/wk, 350mg/kg/wk, 400mg/kg/wk, 450mg/kg/ week and within the range of 500 mg/kg/week. In certain embodiments, the dosage of the bifunctional molecule according to the invention is 10 mg/kg/week to 200 mg/kg/week, 20 mg/kg/week to 150 mg/kg/week, or 25 mg/kg/week. It ranges from week to 100 mg/kg/week. In certain embodiments, the compositions of the invention can be administered for a period of 2 weeks to 6 months, e.g., 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks. Administered 1x weekly for 13 weeks, 26 weeks, 6 months, 8 months, 10 months, or more than 1 year. In certain embodiments, the compositions of the invention are administered 2x per week. In another embodiment, the compositions of the invention are administered every other week.
본 발명의 조성물은 약리학적 유효량의 HAPLN1 단백질 분자 및 약제학적으로 허용되는 담체를 포함하는 약학적 조성물로도 제형화할 수 있다. 약리학적 또는 치료학적 유효량은 의도된 약리학적, 치료적 또는 예방적 결과를 생성하는 데 효과적인 함량을 의미한다. "약리학적 유효량" 및 "치료학적 유효량" 또는 간단히 "유효량"이라는 어구는 의도된 약리학적, 치료적 또는 예방적 결과를 생성하는 데 효과적인 이중작용성 분자의 양을 지칭한다. 예를 들어, 주어진 임상 치료가 질병 또는 장애와 관련된 측정 가능한 매개 변수가 20 % 이상 감소할 때 효과적인 것으로 간주된다면, 그 질병 또는 장애를 치료하기 위한 약물의 치료적 유효량은 해당 매개 변수를 최소 20 % 감소시키기에 필요한 양이다. The composition of the present invention can also be formulated as a pharmaceutical composition containing a pharmacologically effective amount of HAPLN1 protein molecule and a pharmaceutically acceptable carrier. Pharmacologically or therapeutically effective amount means an amount effective to produce the intended pharmacological, therapeutic or prophylactic result. The phrases “pharmacologically effective amount” and “therapeutically effective amount” or simply “effective amount” refer to the amount of a bifunctional molecule that is effective to produce the intended pharmacological, therapeutic or prophylactic result. For example, if a given clinical treatment is considered effective when it reduces a measurable parameter associated with a disease or disorder by at least 20%, then a therapeutically effective dose of a drug to treat that disease or disorder would reduce that parameter by at least 20%. This is the amount needed to reduce it.
본 발명의 적절하게 제형화된 약학적 조성물은 정맥 내, 근육 내, 복강 내, 피하, 경피, 기도 (에어로졸), 직장, 질 및 국소 (협측 및 설하 포함) 투여를 포함하는 비경구 경로와 같은 당해 기술분야에 공지된 임의의 수단에 의해 투여할 수 있다. 일부 구체예에서, 약학적 조성물은 정맥 내 또는 비경구 주입 또는 주사에 의해 투여된다. Suitably formulated pharmaceutical compositions of the invention may be administered by parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, respiratory (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration. Administration may be by any means known in the art. In some embodiments, the pharmaceutical composition is administered by intravenous or parenteral infusion or injection.
일반적으로 분자의 적절한 투여량 단위는 하루당 수용자의 체중 kg 당 0.001 내지 0.25mg의 범위, 또는 1 일당 체중 1kg 당 0.01 내지 20마이크로그램의 범위, 또는 1 일당 체중 kg 당 0.01 내지 10 마이크로그램의 범위, 또는 1 일 체중 1kg 당 0.10 내지 5 마이크로그램의 범위, 또는 1 일당 체중 kg 당 0.1 내지 2.5 마이크로그램의 범위이다. 분자를 포함하는 약학적 조성물은 1 일 1 회 투여할 수 있다. 그러나, 치료제는 또한 하루 종일 적절한 간격으로 투여되는 2, 3, 4, 5, 6 또는 그 이상의 하위 용량을 포함하는 투여 단위로 투여할 수 있다. 투여 단위는 또한, 예를 들어, 수일 기간에 걸쳐 분자의 지속적이고 일관된 방출을 제공하는 통상적인 지속 방출 제형을 사용하여 수일에 걸쳐 단일 용량으로 배합될 수 있다. 지속 방출 제형은 당해 기술분야에 잘 알려져있다. 이 구체예에서, 투여량 단위는 일일 투여량의 상응하는 배수를 포함한다.In general, suitable dosage units of the molecule range from 0.001 to 0.25 mg per kg of body weight of the recipient per day, or from 0.01 to 20 micrograms per kg of body weight per day, or from 0.01 to 10 micrograms per kg of body weight per day, or in the range of 0.10 to 5 micrograms per kilogram of body weight per day, or in the range of 0.1 to 2.5 micrograms per kilogram of body weight per day. A pharmaceutical composition containing the molecule can be administered once daily. However, the therapeutic agent may also be administered in dosage units comprising 2, 3, 4, 5, 6 or more subdoses administered at appropriate intervals throughout the day. Dosage units can also be formulated as single doses over several days, for example, using conventional sustained release formulations that provide sustained and consistent release of the molecule over a period of several days. Sustained release formulations are well known in the art. In this embodiment, the dosage unit comprises a corresponding multiple of the daily dosage.
상기 약학적 조성물은 투여 지침과 함께 키트, 용기, 팩, 또는 디스펜서에 포함될 수 있다.The pharmaceutical composition may be included in a kit, container, pack, or dispenser along with administration instructions.
본 명세서에 사용된 "치료" 또는 "치료하는"은 질병 또는 장애, 질병 또는 장애의 증상, 또는 질병에 대한 소인을 치료, 치유, 완화, 경감, 변경, 구제, 개선, 개량 또는 영향을 주기 위한 목적으로 환자에게 치료제 (예를 들어, 본 발명의 분자)의 적용 또는 투여, 또는 단리된 조직 또는 세포주에 대한 치료제의 적용 또는 투여로 정의되는데, 상기 환자는 질병 또는 장애, 질병 또는 장애의 증상, 또는 질병 또는 장애에 대한 소인을 갖고 있는 것이다.As used herein, “treatment” or “treating” means treating, curing, alleviating, alleviating, altering, relieving, ameliorating, ameliorating, or affecting a disease or disorder, symptoms of a disease or disorder, or predisposition to a disease. It is defined as the application or administration of a therapeutic agent (e.g., a molecule of the invention) to a patient, or the application or administration of a therapeutic agent to an isolated tissue or cell line, for the purpose of treating the patient with a disease or disorder, symptoms of the disease or disorder, Or have a predisposition to disease or disability.
또한 나아가 본 발명의 일 양상은 히알루론산과 프로테오글리칸 연결 단백질 1(hyaluronan and proteoglycan link protein 1; HAPLN1)을 유효성분으로 포함하는 조성물을 세포의 산화작용을 예방 또는 억제에 이용하는 용도에 관한 것이다.Furthermore, one aspect of the present invention relates to the use of a composition containing hyaluronic acid and proteoglycan link protein 1 (HAPLN1) as active ingredients to prevent or inhibit oxidation of cells.
이하 실시예를 통하여 본 발명을 더욱 상세하게 설명하고자 하나, 하기의 실시예는 단지 설명의 목적을 위한 것이며 본원 발명의 범위를 한정하고자 하는 것은 아니다.The present invention will be described in more detail through examples below. However, the examples below are for illustrative purposes only and are not intended to limit the scope of the present invention.
[제조예 1][Production Example 1]
히알루론산과 프로테오글리칸 연결 단백질 1(hyaluronan and proteoglycan link protein 1; HAPLN1)의 제조Preparation of hyaluronan and proteoglycan link protein 1 (HAPLN1)
아미노산 서열번호 1의 인간 HAPLN1 단백질을 코딩하는 폴리뉴클레오티드를 포함하는 벡터를 CHO-K1 세포에 삽입하여 재조합 인간 HAPLN1 단백질을 생산하는 CHO-K1 세포주를 제작하였다. A CHO-K1 cell line producing recombinant human HAPLN1 protein was created by inserting a vector containing a polynucleotide encoding the human HAPLN1 protein of amino acid sequence number 1 into CHO-K1 cells.
상기 아미노산 서열번호 1은 다음과 같다:The amino acid sequence number 1 is as follows:
MKSLLLLVLISICWADHLSDNYTLDHDRAIHIQAENGPHLLVEAEQAKVFSHRGGNVTLPCKFYRDPTAFGSGIHKIRIKWTKLTSDYLKEVDVFVSMGYHKKTYGGYQGRVFLKGGSDSDASLVITDLTLEDYGRYKCEVIEGLEDDTVVVALDLQGVVFPYFPRLGRYNLNFHEAQQACLDQDAVIASFDQLYDAWRGGLDWCNAGWLSDGSVQYPITKPREPCGGQNTVPGVRNYGFWDKDKSRYDVFCFTSNFNGRFYYLIHPTKLTYDEAVQACLNDGAQIAKVGQIFAAWKILGYDRCDAGWLADGSVRYPISRPRRRCSPTEAAVRFVGFPDKKHKLYGVYCFRAYNMKSLLLLVLISICWADHLSDNYTLDHDRAIHIQAENGPHLLLVEAEQAKVFSHRGGNVTLPCKFYRDPTAFGSGIHKIRIKWTKLTSDYLKEVDVFVSMGYHKKTYGGYQGRVFLKGGSDSDASLVITDLTLEDYGRYKCEVIEGLEDDTVVVALDLQGVVFPYFPRLGRYNLNFHEAQQACLDQDAVIASFDQLYDAWRGGLDWCNA GWLSDGSVQYPITKPREPCGGQNTVPGVRNYGFWDKDKSRYDVFCFTSNFNGRFYYLIHPTKLTYDEAVQACLNDGAQIAKVGQIFAAWKILGYDRCDAGWLADGSVRYPISRPRRRCSPTEAAVRFVGFPDKKHKLYGVYCFRAYN
단백질 생산량 및 품질이 우수한 세포주를 MCB(Master Cell Bank)로 선정하였다. 상기 MCB를 계대배양하여 0.40±0.05×106 cells/mL의 농도로 Thermo사의 Hyperforma SUB 250 L 바이오리액터에 접종하고 유가 배양(Fed-Batch Culture) 하였다. 기본 배지로는 22.36 g ActiPro™ 배지 + 0.5846 g 글루타민 + 10.00 g HT Supplement(Thermo Fisher Scientific사) + 4.29 g 10 N NaOH + 1.80 g NaHCO3를 사용하였다. 배양 온도는 36.5℃, 용존산소량(Dissolved oxygen, DO)은 40.0%, pH는 7.00±0.20로 설정하였다. pH 조절 용액으로 1 M 탄산나트륨 일수화물(Sodium Carbonate Monohydrate)을 사용하였다. 공급 배지(feeding medium, FM)로는 181.04 g HyClone™ Cell Boost 7a + 12.28 g 10 N NaOH의 FM020a 및 94.60 g HyClone™ Cell Boost 7b + 105.93 g 10 N NaOH의 FM020b를 사용하였다.Cell lines with excellent protein production and quality were selected as MCB (Master Cell Bank). The MCB was subcultured and inoculated into Thermo's Hyperforma SUB 250 L bioreactor at a concentration of 0.40±0.05×10 6 cells/mL and fed-batch culture was performed. The basic medium used was 22.36 g ActiPro™ medium + 0.5846 g glutamine + 10.00 g HT Supplement (Thermo Fisher Scientific) + 4.29 g 10 N NaOH + 1.80 g NaHCO 3 . The culture temperature was set at 36.5°C, dissolved oxygen (DO) was set at 40.0%, and pH was set at 7.00±0.20. 1 M Sodium Carbonate Monohydrate was used as a pH adjustment solution. As feeding medium (FM), 181.04 g HyClone™ Cell Boost 7a + 12.28 g 10 N NaOH FM020a and 94.60 g HyClone™ Cell Boost 7b + 105.93 g 10 N NaOH FM020b were used.
위와 같이 배양된 세포로부터 재조합 인간 HAPLN1 (rhHAPLN1) 단백질을 회수(Harvest and Clarification), 한외여과/투석여과 1 (Ultrafiltration/Diafiltration 1, UF/DF1), 음이온 교환 크로마토그래피, S/D (Solvent/Detergent) 바이러스 불활성화, 양이온 교환 크로마토그래피, 혼합 모드 크로마토그래피 (Mixed-mode Chromatography, MMC), 소수성 상호작용 크로마토그래피, 한외여과/투석여과 2 (Ultrafiltration/Diafiltration 2, UF/DF2) 및 중간 심층 여과 (Intermediate Depth Filtration, Int. DF) 등의 과정을 거쳐 분리 및 정제하였다. 이하의 실시예에서는 이와 같은 본 발명의 조성물을 이용하였다.Recombinant human HAPLN1 (rhHAPLN1) protein was recovered from cells cultured as above (Harvest and Clarification), Ultrafiltration/Diafiltration 1 (UF/DF1), Anion Exchange Chromatography, S/D (Solvent/Detergent) ) Virus inactivation, cation exchange chromatography, Mixed-mode Chromatography (MMC), hydrophobic interaction chromatography, Ultrafiltration/Diafiltration 2 (UF/DF2) and intermediate depth filtration ( It was separated and purified through processes such as Intermediate Depth Filtration (Int. DF). In the following examples, the composition of the present invention was used.
[제조예 2][Production Example 2]
HAPLN1 단백질을 포함하는 본 발명 조성물의 제조Preparation of compositions of the invention comprising HAPLN1 protein
상기 제조예 1에서 정제한 HAPLN1 단백질을 20 mM 아세트산 완충액, 8% (w/v) 슈크로스, 0.04% (w/v) PS80, pH 5.0에서 안정화시켜 HAPLN1 단백질 자체를 주된 유효성분으로서 포함하는 본 발명의 조성물을 제조하였다.The HAPLN1 protein purified in Preparation Example 1 was stabilized in 20 mM acetic acid buffer, 8% (w/v) sucrose, 0.04% (w/v) PS80, pH 5.0 to produce this product containing the HAPLN1 protein itself as the main active ingredient. The composition of the invention was prepared.
[제조예 3][Production Example 3]
HAPLN1 단백질을 코딩하는 유전자를 담고 있는 발현 벡터를 함유하는 본 발명 조성물의 제조Preparation of a composition of the present invention containing an expression vector containing a gene encoding the HAPLN1 protein
HAPLN1 단백질을 코딩하는 유전자를 담고 있는 발현 벡터를 함유하는 조성물을 jetPRIME Transfection Reagent Kit (PolyPlus사 114-15 제품)을 사용하여 제조하였다. 보다 구체적으로 OriGene사 RC209274 제품을 사용하여 Human HAPLN1을 발현하도록 설계된 플라스미드 벡터를 제조하였다(이하, "Human HAPLN1 ORF Clone"이라 함). 이는 host 세포의 핵 내에서 자신이 수송하는 유전자를 발현시킬 수 있다.A composition containing an expression vector containing the gene encoding the HAPLN1 protein was prepared using the jetPRIME Transfection Reagent Kit (114-15 product from PolyPlus). More specifically, a plasmid vector designed to express Human HAPLN1 was prepared using OriGene's RC209274 product (hereinafter referred to as “Human HAPLN1 ORF Clone”). It can express the genes it transports within the nucleus of the host cell.
참고로 jetPRIME Transfection Reagent Kit는 jetPRIME 완충액과 jetPRIME Transfection 시약으로 구성되며, jetPRIME 완충액은 host 세포로 수송할 플라스미드 벡터를 희석하는 용도이고, jetPRIME Transfection 시약은 플라스미드 벡터를 vesicle(liposome)형태로 포집하여 host 세포 내로 침투되도록 작용한다.For reference, the jetPRIME Transfection Reagent Kit consists of jetPRIME buffer and jetPRIME Transfection reagent. The jetPRIME buffer is used to dilute the plasmid vector to be transported to host cells, and the jetPRIME Transfection reagent captures the plasmid vector in the form of a vesicle (liposome) and transmits it to the host cell. It works to penetrate into the inside.
상기 Human HAPLN1 ORF Clone을 jetPRIME 완충액에 희석하여 HAPLN1 단백질을 코딩하는 유전자를 담고 있는 발현 벡터를 함유하는 본 발명 조성물을 제조하였다.The Human HAPLN1 ORF Clone was diluted in jetPRIME buffer to prepare a composition of the present invention containing an expression vector containing the gene encoding the HAPLN1 protein.
이에 대해 jetPRIME Transfection 시약을 첨가하여 10분간 방치하여 플라스미드 벡터가 vesicle(liposome)형태로 포집되도록 한 후 배양 중인 세포 위에 떨어뜨린다. 세포를 48시간 이상 배양한 후, Human HAPLN1 유전자 발현 증가를 Real-time qPCR로 확인하고, Human HAPLN1 단백질 발현 증가를 Western blot으로 확인하였다.In response, jetPRIME Transfection reagent is added and left for 10 minutes to capture the plasmid vector in the form of a vesicle (liposome) and then dropped onto the cells in culture. After culturing the cells for more than 48 hours, the increase in human HAPLN1 gene expression was confirmed by real-time qPCR, and the increase in human HAPLN1 protein expression was confirmed by Western blot.
[실시예 1][Example 1]
인간 피부 섬유아세포에 대한 본 발명의 조성물의 활성 산소의 억제능의 평가Evaluation of the ability of the composition of the present invention to inhibit oxygen radicals on human skin fibroblasts
인간 피부 섬유아세포(Normal Human Dermal Fibroblast, NHDF)를 제조자의 프로토콜에 따라 9 계대배양 후 6 웰 플레이트에 분주(seeding)(1.5 X 105 cells/well)하고 FBM-2(Fibroblast Growth Medium-2, CC-3131, Lonza) 배지 중 37℃ 5% CO2 조건의 인큐베이터에서 24시간 동안 인큐베이트하였다. 이에 대해 IL-1β(10 ng/ml)를 처리하여 24시간 동안 ROS의 과다 생성을 유도하고 본 발명의 조성물(rhHAPLN1)을 각각 0, 2.5, 5, 10 ng/ml로 처리한 후 24시간 동안 인큐베이트하였다. 이후 DCF-DA(20mM)를 1:20,000 비율로 희석하여 각 세포 샘플에 처리하고 10분간 인큐베이트하였다. 이후 각각의 샘플을 인산완충식염수(phosphate buffered saline ; PBS, pH 7.2)로 2회 세척한 후 트립신으로 각 well의 세포를 회수(harvest)하고 FACS(Fluorescence-Activated Cell Sorting)(BD FACSLyricTM, BD Biosciences)로 세포의 형광을 측정하여 본 발명의 조성물의 함량이 증가함에 따라 ROS 수준의 변화를 관찰하였다. Normal Human Dermal Fibroblast (NHDF) cells were subcultured for 9 times according to the manufacturer's protocol, then seeded ( 1.5 CC-3131, Lonza) medium was incubated for 24 hours in an incubator at 37°C and 5% CO 2 conditions. In response, treatment with IL-1β (10 ng/ml) induced excessive production of ROS for 24 hours, and treatment with the composition of the present invention (rhHAPLN1) at 0, 2.5, 5, and 10 ng/ml, respectively, for 24 hours. Incubated. Afterwards, DCF-DA (20mM) was diluted at a ratio of 1:20,000, treated with each cell sample, and incubated for 10 minutes. Afterwards, each sample was washed twice with phosphate buffered saline (PBS, pH 7.2), cells in each well were harvested with trypsin, and FACS (Fluorescence-Activated Cell Sorting) (BD FACSLyric TM , BD) was performed. Biosciences) measured the fluorescence of cells to observe changes in ROS levels as the content of the composition of the present invention increased.
이의 결과를 나타낸 도 1에 따르면 본 발명의 조성물의 함량이 0, 2.5, 5, 10 ng/ml로 증가함에 따라 IL-1β 처리에 의해 과도하게 생성된 ROS의 수준이 유의하게 감소되는 것을 알 수 있다. 특히 도 1의 C에 따르면 본 발명의 조성물을 처리하지 않은 샘플에 비하여 본 발명의 조성물(rhHAPLN1)을 10 ng/ml로 처리한 후 ROS의 수준이 거의 10배나 감소한 것으로 나타났다.According to Figure 1 showing the results, it can be seen that as the content of the composition of the present invention increases to 0, 2.5, 5, and 10 ng/ml, the level of ROS excessively generated by IL-1β treatment is significantly reduced. there is. In particular, according to Figure 1C, the level of ROS was reduced by almost 10 times after treatment with 10 ng/ml of the composition of the present invention (rhHAPLN1) compared to the sample not treated with the composition of the present invention.
[실시예 2][Example 2]
인간 피부 섬유아세포에 대한 본 발명의 조성물의 항산화 지표의 확인Confirmation of antioxidant index of the composition of the present invention on human skin fibroblasts
인간 피부 섬유아세포(NHDF)를 제조자의 프로토콜에 따라 6 계대 배양 후 60 φ 디쉬에 분주(5 X 105 cells/well)하고 FBM-2(Fibroblast Growth Medium-2, CC-3131, Lonza) 배지로 37℃ 5% CO2 조건의 인큐베이터에서 24시간 동안 인큐베이트하였다. 이에 대해 아무런 처리를 하지 않은 정상군과 IL-1β(10 ng/ml)를 24시간 동안 처리하여 ROS의 과다 생성을 유도한 군으로 나누고 각각에 대해 본 발명의 조성물(rhHAPLN1)을 각각 0, 2.5, 5, 10 ng/ml로 처리한 후 24시간 동안 인큐베이트하였다. 이후 DCF-DA를 가하고 Multi-Mode Reader(488/520nm, Synergy|HTX)로 ROS를 측정하고(도 2의 A), 이후 각각의 샘플을 인산완충식염수(phosphate buffered saline; PBS, pH 7.2)로 2회 세척한 후 단백질을 추출하고 웨스턴 블롯으로 NRF2의 발현 정도를 확인하였다(도 2의 B).Human dermal fibroblasts (NHDF) were cultured for 6 passages according to the manufacturer's protocol, then dispensed into 60 ϕ dishes ( 5 It was incubated for 24 hours in an incubator under 37°C and 5% CO 2 conditions. The group was divided into a normal group without any treatment and a group treated with IL-1β (10 ng/ml) for 24 hours to induce excessive production of ROS, and the composition of the present invention (rhHAPLN1) was administered to each group at a concentration of 0 and 2.5, respectively. , 5, and 10 ng/ml and then incubated for 24 hours. Afterwards, DCF-DA was added and ROS was measured with a Multi-Mode Reader (488/520nm, Synergy|HTX) (A in Figure 2), and then each sample was washed with phosphate buffered saline (PBS, pH 7.2). After washing twice, the protein was extracted, and the expression level of NRF2 was confirmed by Western blot (Figure 2B).
이의 결과를 나타낸 도 2에 따르면 본 발명의 조성물의 함량이 0, 2.5, 5, 10 ng/ml로 증가함에 따라 DCF-DA에 의한 형광 강도가 단계적으로 감소되는 것으로 보아 IL-1β 처리에 의해 과도하게 생성된 ROS의 수준이 단계적으로 유의하게 감소되는 것을 알 수 있다. 즉 도 2의 A에 따르면 본 발명의 조성물을 처리하지 않은 샘플(0 ng/ml)에 비하여 본 발명의 조성물(rhHAPLN1)을 10 ng/ml로 처리한 후 ROS의 수준이 약 2/3로 감소한 것으로 나타났다. 또한 도 2의 B에 따르면 본 발명의 조성물을 처리하지 않은 샘플(0 ng/ml)에 비하여 본 발명의 조성물(rhHAPLN1)을 10 ng/ml로 처리한 후 항산화 마커인 NRF2의 발현량이 확연히 증가된 것으로 나타나 항산화 반응이 활성화되었다는 점을 알 수 있다.According to Figure 2, which shows the results, as the content of the composition of the present invention increases to 0, 2.5, 5, and 10 ng/ml, the fluorescence intensity by DCF-DA decreases stepwise, indicating that it is excessive due to IL-1β treatment. It can be seen that the level of ROS generated is significantly reduced step by step. That is, according to Figure 2A, the level of ROS was reduced by about 2/3 after treatment with the composition of the present invention (rhHAPLN1) at 10 ng/ml compared to the sample (0 ng/ml) not treated with the composition of the present invention. It was found that In addition, according to Figure 2B, the expression level of NRF2, an antioxidant marker, was significantly increased after treatment with the composition of the present invention (rhHAPLN1) at 10 ng/ml compared to the sample (0 ng/ml) that was not treated with the composition of the present invention. It can be seen that the antioxidant reaction was activated.
[실시예 3][Example 3]
인간 폐 섬유아세포에 대한 본 발명의 조성물의 활성 산소의 억제능의 평가Evaluation of the reactive oxygen species inhibition ability of the composition of the present invention on human lung fibroblasts
인간 폐 섬유아세포(Normal Human Lung Fibroblast, NHLF)를 96 웰 플레이트에 분주(8 X 103 cells/well)하고 24시간 동안 인큐베이트하였다. 이에 대해 IL-1β(10 ng/ml)를 24시간 동안 처리하여 ROS의 과다 생성을 유도한 군과 IL-1β(10 ng/ml)를 처리하지 않은 군에 대해 본 발명의 조성물(rhHAPLN1)을 각각 0, 5 ng/ml로 첨가한 후 24시간 동안 인큐베이트하였다. 이후 DCF-DA(20mM)를 1:20,000 비율로 희석하여 각 세포 샘플에 처리하고 10분간 인큐베이트하였다. 이후 각각의 샘플을 Multi-Mode Reader(492/530nm, Synergy|HTX)로 활성 산소(ROS)의 상대적인 생성 형광 정도를 측정하였다.Normal Human Lung Fibroblast (NHLF) was distributed in a 96 well plate (8 × 10 3 cells/well) and incubated for 24 hours. In this regard, the composition of the present invention (rhHAPLN1) was administered to the group treated with IL-1β (10 ng/ml) for 24 hours to induce excessive production of ROS and the group not treated with IL-1β (10 ng/ml). They were added at 0 and 5 ng/ml, respectively, and incubated for 24 hours. Afterwards, DCF-DA (20mM) was diluted at a ratio of 1:20,000, treated with each cell sample, and incubated for 10 minutes. Afterwards, the relative fluorescence level of reactive oxygen species (ROS) was measured for each sample using a Multi-Mode Reader (492/530nm, Synergy|HTX).
이의 결과를 나타낸 도 3에 따르면 본 발명의 조성물의 함량 5 ng/ml로 처리한 경우 IL-1β 처리에 의해 과도하게 생성된 ROS의 수준이 유의하게 감소된 것을 알 수 있다.According to Figure 3 showing the results, it can be seen that when treated with the composition of the present invention at an amount of 5 ng/ml, the level of ROS excessively generated by IL-1β treatment was significantly reduced.
[실시예 4][Example 4]
인간 폐 섬유아세포에 대한 본 발명의 조성물의 항산화 지표의 확인Confirmation of antioxidant index of the composition of the present invention on human lung fibroblasts
인간 폐 섬유아세포(NHLF)를 제조자의 프로토콜에 따라 6 계대 배양 후 100 φ 디쉬에 분주(6.5 X 105 cells/well)하고 FBM-2(Fibroblast Growth Medium-2, CC-3131, Lonza) 배지로 37℃ 5% CO2 조건의 인큐베이터에서 24시간 동안 인큐베이트하였다. 이에 대해 IL-1β(10 ng/ml)를 처리하여 ROS의 과다 생성을 유도한 군과 아무런 처리를 하지 않은 정상군으로 나누고 각각에 대해 본 발명의 조성물(rhHAPLN1)을 각각 0, 5 ng/ml로 처리한 후 24시간 동안 인큐베이트하였다. 이후 RIPA 완충액(Radioimmunoprecipitation assay buffer)과 프로테아제/포스파타제 inhibitor cocktail로 세포를 용해 후 전체 단백질 추출물은 10% 아크릴아미드 겔 상에서 SDS-PAGE로 분리하였다.Human lung fibroblasts (NHLF) were cultured for 6 passages according to the manufacturer's protocol, then dispensed into 100 ϕ dishes (6.5 It was incubated for 24 hours in an incubator under 37°C and 5% CO 2 conditions. In this regard, the group was divided into a group treated with IL-1β (10 ng/ml) to induce excessive production of ROS and a normal group without any treatment, and the composition of the present invention (rhHAPLN1) was administered to each group at 0 and 5 ng/ml, respectively. After treatment, it was incubated for 24 hours. Afterwards, cells were lysed with RIPA buffer (Radioimmunoprecipitation assay buffer) and protease/phosphatase inhibitor cocktail, and the total protein extract was separated by SDS-PAGE on a 10% acrylamide gel.
이와 같은 샘플들에 대해 면역블롯을 위해 세포 추출물(cell lysate)을 10 μg 로딩하고, 면역검출을 위해 NRF2에 대한 1차 항체(5% BSA-TBST(Bovine Serum Albumin-Tris-Buffered Saline & Polysorbate 20)에서 1:1,000 희석)로 4℃에서 밤새 진탕하였다. HRP(Horseradish Peroxidase)가 컨쥬게이트된 2차 항체(5% skim milk-TBST에서 1:10,000 희석)로 실온에서 1시간동안 인큐베이트하고 단백질 밴드는 ECL(Enhanced Chemiluminenscent, Femto) 용액으로 현상 후 FUSION FX(Vilber)로 확인하였다.For these samples, 10 μg of cell extract (cell lysate) was loaded for immunoblot, and primary antibody against NRF2 (5% BSA-TBST (Bovine Serum Albumin-Tris-Buffered Saline & Polysorbate 20) was used for immunodetection. ) at a 1:1,000 dilution) and shaken overnight at 4°C. Incubate for 1 hour at room temperature with HRP (Horseradish Peroxidase)-conjugated secondary antibody (diluted 1:10,000 in 5% skim milk-TBST), and develop the protein band with ECL (Enhanced Chemiluminenscent, Femto) solution and then FUSION FX. (Vilber).
이의 결과를 나타낸 도 4에 따르면 IL-1β로 활성 산소(ROS)의 과다생성을 유도한 조건에서는 항산화 관련 단백질인 NRF2의 발현량이 매우 희박했지만 이에 대해 본 발명의 조성물(rhHAPLN1)을 5 ng/ml 처리한 경우에는 NRF2의 발현량이 정상세포 조건의 경우와 거의 동일한 것을 NRF2의 발현 밴드의 진하기와 두께로부터 확인 가능하다.According to Figure 4 showing the results, under conditions in which excessive production of reactive oxygen species (ROS) was induced by IL-1β, the expression level of NRF2, an antioxidant-related protein, was very low, but the composition of the present invention (rhHAPLN1) was used at 5 ng/ml. In the case of treatment, it can be confirmed from the intensity and thickness of the NRF2 expression band that the expression level of NRF2 is almost the same as that in normal cell conditions.
[실시예 5][Example 5]
인간 각막 상피세포에 대한 본 발명의 조성물의 활성 산소의 억제능의 평가 1Evaluation of the reactive oxygen species inhibitory ability of the composition of the present invention on human corneal epithelial cells 1
인간 각막 상피세포(Human Corneal Epithelial Cell, HCEC)를 제조사의 프로토콜에 따라 5 계대 배양 후 96 웰 플레이트에 분주(1.6 X 104 cells/well)하고 Corneal Epithelial Cell Basal Medium (ATCC PCS-700-030, ATCC) 배지를 사용하여 37℃ 5% CO2 조건의 인큐베이터에서 24시간 동안 인큐베이트하였다. 이에 대해 5M NaCl로 450mOsM 배지를 제조하여 처리하고, 48시간 동안 인큐베이트하였다. 참고로 본 실시예에서 314mOsM의 경우 정상 대조군으로 보되, 상대적으로 높은 삼투압인 450mOsM의 경우는 안구건조증 모델로 설정할 수 있을 정도로 활성 산소의 발생을 유발하는 조건이다.Human Corneal Epithelial Cell (HCEC) were subcultured for 5 times according to the manufacturer's protocol , then dispensed into 96 well plates (1.6 ATCC) medium was used and incubated for 24 hours in an incubator at 37°C and 5% CO 2 conditions. For this, 450mOsM medium was prepared and treated with 5M NaCl, and incubated for 48 hours. For reference, in this example, 314 mOsM is considered a normal control, but the relatively high osmotic pressure of 450 mOsM is a condition that induces the generation of active oxygen enough to be set as a dry eye syndrome model.
보다 구체적으로, 314mOsM 배지에 세포를 분주(seeding)하고 24시간 뒤에 활성 산소 발생의 유발조건인 450mOsM 배지로 교체하고 활성 산소 발생의 유발 조건(450mOsM 배지) 하의 각 샘플에 대해 본 발명의 조성물(rhHAPLN1)을 각각 0, 5, 10, 20, 50, 100 ng/ml 함량으로 처리한 후, 24시간 동안 인큐베이트하였다. 배지를 제거한 후 각각의 샘플을 인산완충식염수(phosphate buffered saline; PBS, pH 7.2)로 2회 세척한 후 DCF-DA 용액(20mM/ml)을 배지에 희석하여 25μM로 만들고, well 당 100λ씩 처리하고 45분간 인큐베이트하였다.More specifically, cells were seeded in 314mOsM medium, and 24 hours later, the medium was replaced with 450mOsM medium, which is a condition for inducing active oxygen generation. For each sample under the condition for inducing active oxygen generation (450mOsM medium), the composition of the present invention (rhHAPLN1 ) were treated at concentrations of 0, 5, 10, 20, 50, and 100 ng/ml, respectively, and then incubated for 24 hours. After removing the medium, each sample was washed twice with phosphate buffered saline (PBS, pH 7.2), then the DCF-DA solution (20mM/ml) was diluted in the medium to make 25μM, and treated with 100λ per well. and incubated for 45 minutes.
이에 대해 인산완충식염수로 재차 2회 세척한 후 Multi-Mode Reader(485/528nm, Synergy|HTX)를 사용하여 활성 산소가 발생시키는 형광의 정도를 검출하였다.After washing it twice again with phosphate-buffered saline solution, the degree of fluorescence generated by active oxygen was detected using a Multi-Mode Reader (485/528nm, Synergy|HTX).
이의 결과를 나타낸 도 5에 따르면 본 발명의 조성물의 함량이 0, 5, 10, 20, 50, 100 ng/ml로 증가함에 따라 고삼투압(450mOsM 배지) 처리에 의해 과도하게 생성된 ROS의 수준이 유의하게 감소되는 것을 알 수 있다. 특히 본 발명의 조성물을 처리하지 않은 샘플에 비하여 본 발명의 조성물(rhHAPLN1)을 20 내지 50ng/ml로 처리한 경우 ROS의 수준이 가장 감소한 것으로 나타났다.According to Figure 5, which shows the results, as the content of the composition of the present invention increases to 0, 5, 10, 20, 50, and 100 ng/ml, the level of ROS excessively generated by treatment with high osmotic pressure (450 mOsM medium) increases. It can be seen that there is a significant decrease. In particular, compared to samples not treated with the composition of the present invention, the level of ROS was found to be most reduced when treated with the composition of the present invention (rhHAPLN1) at 20 to 50 ng/ml.
[실시예 6][Example 6]
인간 각막 상피세포에 대한 본 발명의 조성물의 항산화 지표의 확인Confirmation of antioxidant index of the composition of the present invention for human corneal epithelial cells
인간 각막 상피세포(HCEC)를 5 계대 배양 후 100mm 디쉬에 분주(seeding)(3.8 X 105 cells/100mm dish)하고 24시간 인큐베이트한 후 450mOsM 배지로 처리하였다. 참고로 여기서 450mOsM 배지는 기존의 314mOsM에 5M NaCl를 처리(100ml 기준 5M NaCl 1.424ml)함으로써 제작하였다.Human corneal epithelial cells (HCEC) were cultured for 5 passages, seeded on 100mm dishes (3.8 For reference, here, the 450mOsM medium was produced by treating the existing 314mOsM with 5M NaCl (1.424ml of 5M NaCl per 100ml).
450mOsM 배지 처리 48시간 후 본 발명의 조성물(rhHAPLN1)(0, 10, 100 ng/ml)을 배지에 첨가한 후 24시간 동안 인큐베이트했다. 이후 RIPA 버퍼액과 프로테아제/포스파타제 inhibitor cocktail로 세포를 용해 후 전체 단백질 추출물을 추출하고 NRF2, GPx1, SOD1, GAPDH의 발현 확인을 위해 단백질을 well당 각각 20μg으로 로딩하고, 4-15% 구배 아크릴아미드 겔 상에서 SDS-PAGE로 분리하였다. 이와 같은 샘플들에 대해 면역검출을 위해 1차 항체(1% BSA-TBST에서 1:1,000 희석)로 4℃에서 밤새 진탕하였다. HRP가 컨쥬게이트된 2차 항체(1% BSA-TBST에서 1:2,000 희석)로 실온에서 1시간동안 인큐베이트하고 단백질 밴드를 ECL(Femto) 용액으로 현상 후 FUSION FX(Vilber)로 확인하였다.After 48 hours of treatment with 450mOsM medium, the composition of the present invention (rhHAPLN1) (0, 10, 100 ng/ml) was added to the medium and incubated for 24 hours. Afterwards, the cells were lysed with RIPA buffer and protease/phosphatase inhibitor cocktail, the total protein extract was extracted, and proteins were loaded at 20 μg per well to confirm the expression of NRF2, GPx1, SOD1, and GAPDH, and 4-15% gradient acrylamide. Separated by SDS-PAGE on gel. These samples were shaken overnight at 4°C with primary antibody (1:1,000 dilution in 1% BSA-TBST) for immunodetection. It was incubated with HRP-conjugated secondary antibody (1:2,000 dilution in 1% BSA-TBST) at room temperature for 1 hour, and the protein band was developed with ECL (Femto) solution and confirmed with FUSION FX (Vilber).
이의 결과를 나타낸 도 6에 따르면 정상 조건(314 mOsM) 및 활성 산소(ROS)의 과다생성을 유도한 조건(450 mOsM) 각각에 있어서, 정상 인간 각막 상피세포에 대해 본 발명의 조성물(rhHAPLN1)을 각각 10, 100 ng/ml로 처리한 후 24시간 인큐베이트한 시점에서 항산화 작용의 지표가 되는 NRF2, GPx1, SOD1 단백질들이 본 발명의 조성물(rhHAPLN1)의 함량에 비례하여 정상 조건의 세포에 필적할 정도로 발현된 것으로 보아 본 발명의 조성물은 고삼투압 조건에서 과도하게 생성된 ROS를 유의적으로 감소시키는 효과가 있음을 알 수 있다.According to Figure 6, which shows the results, the composition of the present invention (rhHAPLN1) was applied to normal human corneal epithelial cells under normal conditions (314 mOsM) and conditions that induced excessive production of reactive oxygen species (ROS) (450 mOsM). After treatment with 10 and 100 ng/ml, respectively, and incubation for 24 hours, NRF2, GPx1, and SOD1 proteins, which are indicators of antioxidant activity, were comparable to cells under normal conditions in proportion to the content of the composition of the present invention (rhHAPLN1). Judging by the degree of expression, it can be seen that the composition of the present invention has the effect of significantly reducing ROS excessively generated under high osmotic pressure conditions.
[실시예 7][Example 7]
외부 유입의 ROS에 대한 인간 폐 미세혈관 내피세포에 대한 본 발명의 조성물의 활성 산소를 차단하는 능력의 평가Evaluation of the ability of the composition of the present invention to block free radicals against externally introduced ROS on human lung microvascular endothelial cells.
인간 폐 미세혈관 내피세포(Human Lung Microvascular Endothelial Cell, HMVEC-L)를 96 웰 플레이트에 분주(seeding)(1000 cells/well)하고 EGMTM-2 완전 배지에서 37℃, 5% CO2 조건으로 24시간 동안 인큐베이트하였다. 이에 대해 본 발명의 조성물(rhHAPLN1) 30 ng/ml을 2시간 동안 처리한 군과 처리하지 않은 군으로 나누었다. 이후 세포를 DCF-DA 20μM로 20분간 인큐베이트하였다. 각 샘플에 대해 250μM로 외부로부터의 ROS로서 H2O2를 가하였다. 여기파장 485nm 및 방출 파장 535nm로써 형광 광도계(Synergy HTX Multi-Mode Reader, BioTek Instruments)로 형광 강도를 검출하였다.Human Lung Microvascular Endothelial Cell (HMVEC-L) were seeded (1000 cells/well) in a 96 well plate and cultured in EGM TM -2 complete medium at 37°C and 5% CO 2 for 24 hours. Incubated for an hour. In this regard, the groups were divided into a group treated with 30 ng/ml of the composition of the present invention (rhHAPLN1) for 2 hours and a group not treated. Afterwards, the cells were incubated with 20 μM DCF-DA for 20 minutes. H 2 O 2 was added as an external ROS at 250 μM to each sample. Fluorescence intensity was detected with a fluorescence photometer (Synergy HTX Multi-Mode Reader, BioTek Instruments) with an excitation wavelength of 485 nm and an emission wavelength of 535 nm.
이의 결과를 나타낸 도 7에 따르면 본 발명의 조성물을 가한 후 시간이 지남에 따라 비례적으로 외부의 ROS 유입(H2O2)에 의해 과도하게 생성된 ROS의 수준이 유의하게 감소되는 것을 알 수 있다. 특히 본 발명의 조성물은 물론 H2O2도 가하지 않은 샘플인 대조군을 기준점으로 볼 경우 본 발명의 조성물을 처리하지 않은 샘플에 비하여 본 발명의 조성물(rhHAPLN1)을 처리한 샘플은 처리 후 30분에 ROS의 수준이 약 1/6로 감소한 것으로 나타났다. 이는 외부로부터 유입되는 ROS에 대해서도 본 발명의 조성물은 효과적으로 차단하여 항산화 작용을 예방적으로 실현할 수 있다는 것을 강력히 시사하는 것이다.According to Figure 7 showing the results, it can be seen that the level of ROS excessively generated by external ROS influx (H 2 O 2 ) is significantly reduced proportionally over time after adding the composition of the present invention. there is. In particular, when considering the control group, which is a sample to which neither the composition of the present invention nor H 2 O 2 was added, as a reference point, the sample treated with the composition of the present invention (rhHAPLN1) compared to the sample not treated with the composition of the present invention 30 minutes after treatment It was found that the level of ROS was reduced by about 1/6. This strongly suggests that the composition of the present invention can effectively block ROS flowing in from the outside and achieve preventive antioxidant action.
[실시예 8] [Example 8]
외부 유입의 ROS에 대한 인간 폐 미세혈관 내피세포에 대한 본 발명의 조성물의, 활성 산소 차단 지표의 확인Identification of the active oxygen blocking index of the composition of the present invention on human lung microvascular endothelial cells against external inflow of ROS
인간 폐 미세혈관 내피세포(HMVEC-L)를 T25 세포 배양 플라스크에 분주(seeding)(50000 cells/well)하고 EGMTM-2 완전 배지에서 37℃, 5% CO2 조건으로 24시간 동안 인큐베이트하였다. 이에 대해 본 발명의 조성물(rhHAPLN1) 30 ng/ml를 2시간 동안 처리한 군과 처리하지 않은 군으로 나누었다. 각 샘플에 대해 250μM로 외부로부터의 ROS로서 H2O2를 가하고 15분(도 8a), 2시간(도 8b) 동안 인큐베이트하였다. 지정된 시간 이후 웨스턴블롯으로 ROS에 대한 응답반응을 나타내는 지표가 되는 단백질 마커들인 pAkt, NRF2, pVEGFR2(Y1175) 및 VEGFR2와 발현 검증 마커인 GAPDH의 발현량을 검출하였다. Human lung microvascular endothelial cells (HMVEC-L) were seeded (50,000 cells/well) in a T25 cell culture flask and incubated in EGM TM -2 complete medium at 37°C and 5% CO 2 for 24 hours. . In this regard, the groups were divided into a group treated with 30 ng/ml of the composition of the present invention (rhHAPLN1) for 2 hours and a group not treated. For each sample, H 2 O 2 was added as external ROS at 250 μM and incubated for 15 minutes (Figure 8a) and 2 hours (Figure 8b). After the specified time, the expression level of protein markers pAkt, NRF2, pVEGFR2 (Y1175), and VEGFR2, which are indicators of response to ROS, and GAPDH, an expression verification marker, were detected by Western blot.
일반적으로 외부로부터의 ROS는 세포 내로 진입하면 Akt를 인산화(pAkt)시키고 이는 NRF2의 발현을 증가시키는데 이는 ROS에 대한 응답반응(response)이다. 그렇다면, pAkt 및 NRF2의 발현 수준은 세포 내로 진입하는 ROS의 수준을 반영할 수 있다. 즉 외부의 ROS가 세포내로 진입할 경우 PI3K/Akt를 통하여 NRF2를 활성화하게 되고 이와 같이 NRF2가 활성화되면 전사인자로서 세포핵으로 진입하여 여러가지 항산화 효소(anti-oxidation enzyme)를 전사시킨다. 여기서 항산화 효소의 발현은 수 시간이 걸리는 과정으로서 결과적으로는 세포내로 진입한 ROS가 세포의 산화를 유도하게 한다.Generally, when ROS from outside enters the cell, it phosphorylates Akt (pAkt), which increases the expression of NRF2, which is a response to ROS. If so, the expression levels of pAkt and NRF2 may reflect the levels of ROS entering the cell. That is, when external ROS enters the cell, it activates NRF2 through PI3K/Akt. When NRF2 is activated, it enters the cell nucleus as a transcription factor and transcribes various anti-oxidation enzymes. Here, the expression of antioxidant enzymes is a process that takes several hours, and ultimately causes ROS entering the cells to induce oxidation of the cells.
본 실험 결과에 따르면 인체는 ROS가 침투할 경우 이의 제거를 위해 자연스레 Akt의 인산화를 통해 특히 항산화 관련 전사인자(NRF2)를 활성화시킨다는 것을 알 수 있다(도 8a에서 중간 레인). 그러나 본 발명의 조성물(rhHAPLN1)을 전처리(30 ng/mL)한 경우에는 H2O2처리 15분 후에 pAkt 및 NRF2의 발현 수준이 본 발명의 조성물로 전처리하지 않은 경우에 비해 그다지 증가하지 않는다(도 8a에서 우측 레인). 여기서 본 발명의 조성물을 전처리한 경우에 pAkt 및 NRF2의 발현 수준이 증가하지 않았다는 것은 세포 내로 진입한 ROS의 양이 적다는 의미이다. 이는 본 발명의 조성물이 외부 ROS의 세포 내 진입을 차단하는 효과에 있어서 자연상태의 경우보다 훨씬 뛰어난 것을 나타내는 것이다. 물론 이에 따라 세포의 산화를 유도하는 정도도 낮아지게 될 것이다.According to the results of this experiment, the human body naturally activates the antioxidant-related transcription factor (NRF2) through phosphorylation of Akt to remove ROS when it infiltrates (middle lane in Figure 8a). However, when pretreated (30 ng/mL) with the composition of the present invention (rhHAPLN1), the expression levels of pAkt and NRF2 did not increase significantly after 15 minutes of H 2 O 2 treatment compared to the case where the composition of the present invention was not pretreated ( right lane in Figure 8A). Here, the fact that the expression levels of pAkt and NRF2 did not increase when pretreated with the composition of the present invention means that the amount of ROS entering the cells was small. This indicates that the composition of the present invention is much more effective than the natural state in blocking the entry of external ROS into cells. Of course, this will also lower the degree to which oxidation of cells is induced.
한편, VEGFR2는 내피 세포의 증식, 생존 및 혈관 생성의 마커로서 1175번 아미노산인 티로신(tyrosine)이 인산화되면 분해가 시작된다. 그러나 본 발명의 조성물(rhHAPLN1)은 이러한 인산화를 억제하여 VEGFR2의 세포표면 제시량을 증가시킴으로써 혈관 내 내피세포에서 증식, 생존 및 혈관생성을 위한 신호를 촉발할 수 있게 된다고 해석할 수 있다. 즉 도 8b에 따르면 ROS로서 H2O2처리 2시간 후에는 VEGFR2의 발현량이 ROS가 없는 자연상태의 경우에 비하여 상당히 떨어지지만 본 발명의 조성물(rhHAPLN1)을 전처리한 경우에는 그 발현량이 상당히 회복된 것으로 나타난다. Meanwhile, VEGFR2 is a marker of endothelial cell proliferation, survival, and angiogenesis, and its decomposition begins when tyrosine, amino acid 1175, is phosphorylated. However, it can be interpreted that the composition of the present invention (rhHAPLN1) inhibits this phosphorylation and increases the amount of VEGFR2 displayed on the cell surface, thereby triggering signals for proliferation, survival, and angiogenesis in endothelial cells within blood vessels. That is, according to Figure 8b, after 2 hours of treatment with H 2 O 2 as ROS, the expression level of VEGFR2 is significantly lower than in the natural state without ROS, but when pretreated with the composition of the present invention (rhHAPLN1), the expression level is significantly recovered. It appears that
결과적으로 위 실험결과를 통하여 외부로부터 유입되는 ROS에 대해 본 발명의 조성물이 항산화 작용을 나타내는 메커니즘을 가늠할 수 있다.As a result, through the above experimental results, it is possible to estimate the mechanism by which the composition of the present invention exhibits an antioxidant effect against ROS introduced from the outside.
[실시예 9][Example 9]
외부 유입의 ROS에 대한 인간 폐 미세혈관 내피세포에 대한 본 발명의 조성물의, 활성 산소에 의한 세포증식 억제를 방지하는 능력의 확인Confirmation of the ability of the composition of the present invention to prevent inhibition of cell proliferation by active oxygen in human lung microvascular endothelial cells against external ROS.
인간 폐 미세혈관 내피세포(HMVEC-L)를 96 웰 배양 플레이트에 분주(seeding)(1500 cells/well)하고 EGMTM-2 완전 배지에서 37℃, 5% CO2 조건으로 24시간 동안 인큐베이트하였다. 이에 대해 본 발명의 조성물(rhHAPLN1)을 일정한 농도(3, 10, 30 ng/ml)로 2시간 동안 처리하였다. 세포 샘플들에 대해 H2O2를 1mM로 가하고 24시간(도 9의 A) 또는 48시간(도 9의 B) 동안 인큐베이트하였다. 세포의 증식은 Cell Counting kit-8 (Dojindo)로 평가하였다.Human lung microvascular endothelial cells (HMVEC-L) were seeded (1500 cells/well) in a 96 well culture plate and incubated in EGM TM -2 complete medium at 37°C and 5% CO 2 for 24 hours. . In response, the composition of the present invention (rhHAPLN1) was treated at constant concentrations (3, 10, 30 ng/ml) for 2 hours. H 2 O 2 was added to the cell samples at 1mM and incubated for 24 hours (A in FIG. 9) or 48 hours (B in FIG. 9). Cell proliferation was assessed using Cell Counting kit-8 (Dojindo).
도 9는 세포 외부로부터 직접 활성 산소(ROS)가 유입되었을 때 본 발명 조성물의 투여량에 따른 본 발명의 조성물(rhHAPLN1)에 의한 세포 증식율의 변화를 나타낸다. 즉 인간 폐 미세혈관 내피세포에 대해 본 발명의 조성물(rhHAPLN1)을 0, 3, 10, 및 30 ng/ml을 첨가할 경우 본 발명의 조성물을 처리하지 않은 경우에 비해 세포의 증식률이 훨씬 증가된 것을 알 수 있다. 특히 48시간이 지난 후에 본 발명의 조성물을 처리하지 않은 경우는 세포의 증식률이 24시간이 지난 후에 비해 절반으로 떨어졌으나 본 발명의 조성물을 3 ng/ml 이상으로 첨가한 경우 그의 2배 정도로 높게 유지되고 있는 것으로 나타난다. 이는 외부로부터 유입되는 ROS가 세포의 증식을 억제하는 작용을 본 발명의 조성물은 효과적으로 방지할 수 있다는 것을 강력히 시사하는 것이다.Figure 9 shows the change in cell proliferation rate by the composition of the present invention (rhHAPLN1) according to the dosage of the composition of the present invention when reactive oxygen species (ROS) are introduced directly from the outside of the cell. That is, the composition of the present invention (rhHAPLN1) was administered to human lung microvascular endothelial cells at 0, 3, 10, and 30. It can be seen that when ng/ml is added, the proliferation rate of cells is significantly increased compared to the case where the composition of the present invention is not treated. In particular, when the composition of the present invention was not treated after 48 hours, the cell proliferation rate was reduced to half compared to after 24 hours, but the composition of the present invention was used for 3 hours. When added at ng/ml or more, it appears to be maintained at about twice that level. This strongly suggests that the composition of the present invention can effectively prevent the action of ROS introduced from the outside to inhibit cell proliferation.
본 발명의 조성물 및 방법에 의하면 세포의 산화 작용을 예방 및 억제함으로써 세포의 과도한 산화 작용이 개입하여 유발하는 각종 질환의 발생 또는 진행을 근본적으로 억제함으로써 관련 질환을 예방 내지 치료할 수 있도록 한다.According to the composition and method of the present invention, by preventing and inhibiting the oxidation action of cells, the occurrence or progression of various diseases caused by excessive oxidation action of cells is fundamentally suppressed, thereby making it possible to prevent or treat related diseases.
또한 본 발명의 시약 조성물은 산화작용과 관련된 각종 질환 연구시에 사용되고 관련 질환의 예방 또는 치료 조성물을 개발하는 데에 도움을 줄 수 있다.Additionally, the reagent composition of the present invention can be used in research on various diseases related to oxidation and can help develop compositions for preventing or treating related diseases.
서열번호 1: SEQ ID NO: 1:
MKSLLLLVLISICWADHLSDNYTLDHDRAIHIQAENGPHLLVEAEQAKVFSHRGGNVTLPCKFYRDPTAFGSGIHKIRIKWTKLTSDYLKEVDVFVSMGYHKKTYGGYQGRVFLKGGSDSDASLVITDLTLEDYGRYKCEVIEGLEDDTVVVALDLQGVVFPYFPRLGRYNLNFHEAQQACLDQDAVIASFDQLYDAWRGGLDWCNAGWLSDGSVQYPITKPREPCGGQNTVPGVRNYGFWDKDKSRYDVFCFTSNFNGRFYYLIHPTKLTYDEAVQACLNDGAQIAKVGQIFAAWKILGYDRCDAGWLADGSVRYPISRPRRRCSPTEAAVRFVGFPDKKHKLYGVYCFRAYNMKSLLLLVLISICWADHLSDNYTLDHDRAIHIQAENGPHLLLVEAEQAKVFSHRGGNVTLPCKFYRDPTAFGSGIHKIRIKWTKLTSDYLKEVDVFVSMGYHKKTYGGYQGRVFLKGGSDSDASLVITDLTLEDYGRYKCEVIEGLEDDTVVVALDLQGVVFPYFPRLGRYNLNFHEAQQACLDQDAVIASFDQLYDAWRGGLDWCNA GWLSDGSVQYPITKPREPCGGQNTVPGVRNYGFWDKDKSRYDVFCFTSNFNGRFYYLIHPTKLTYDEAVQACLNDGAQIAKVGQIFAAWKILGYDRCDAGWLADGSVRYPISRPRRRCSPTEAAVRFVGFPDKKHKLYGVYCFRAYN
Claims (12)
- 히알루론산과 프로테오글리칸 연결 단백질 1(hyaluronan and proteoglycan link protein 1; HAPLN1) 또는 이를 코딩하는 유전자를 유효성분으로 포함하는, 항산화 조성물.An antioxidant composition comprising hyaluronan and proteoglycan link protein 1 (HAPLN1) or a gene encoding the same as an active ingredient.
- 청구항 1에 있어서, 상기 단백질이 서열번호 1의 아미노산 서열에 대해 80% 이상의 서열동일성을 갖는 것인 항산화 조성물.The antioxidant composition of claim 1, wherein the protein has more than 80% sequence identity to the amino acid sequence of SEQ ID NO: 1.
- 청구항 1에 있어서, 상기 유전자에 관한 핵산이 발현 벡터 내에 포함되어 있는 것인 항산화 조성물.The antioxidant composition according to claim 1, wherein the nucleic acid for the gene is contained in an expression vector.
- 청구항 1에 있어서, 상기 조성물이 세포의 산화작용을 예방 또는 억제하는 하는 것인 항산화 조성물.The antioxidant composition according to claim 1, wherein the composition prevents or inhibits oxidation of cells.
- 청구항 1에 있어서, 상기 조성물이 세포에서의 활성 산소의 과도한 발생을 억제하거나 이미 발생된 활성 산소를 감소시키는 것인 항산화 조성물.The antioxidant composition according to claim 1, wherein the composition suppresses excessive generation of active oxygen in cells or reduces active oxygen already generated.
- 청구항 4에 있어서, 상기 세포가 섬유아세포, 상피세포 및 내피세포로 이루어진 군에서 선택된 것인 항산화 조성물.The antioxidant composition according to claim 4, wherein the cells are selected from the group consisting of fibroblasts, epithelial cells, and endothelial cells.
- 청구항 1에 있어서, 상기 세포가 in vitro 상태의 세포인 것인 항산화 조성물.The antioxidant composition according to claim 1, wherein the cells are in vitro cells.
- 청구항 1에 있어서, 상기 상기 조성물의 세포에 대한 1회 투여량이 0.1ng/ml 내지 1000ng/ml인 것인 조성물The composition of claim 1, wherein a single dose of the composition to cells is 0.1 ng/ml to 1000 ng/ml.
- 청구항 1에 있어서, 상기 조성물이 세포의 산화작용을 예방 또는 억제하기 위한 조성물의 주요 또는 보조 유효성분으로서 포함되는 것인 항산화 조성물.The antioxidant composition according to claim 1, wherein the composition is included as a main or auxiliary active ingredient in a composition for preventing or inhibiting oxidation of cells.
- 히알루론산과 프로테오글리칸 연결 단백질 1(hyaluronan and proteoglycan link protein 1; HAPLN1)의 단백질 또는 이를 코딩하는 유전자를 유효성분으로 포함하는 조성물을 세포에 처리함으로써 세포의 산화작용을 예방 또는 억제하는 방법.A method of preventing or inhibiting oxidation of cells by treating cells with a composition containing the hyaluronan and proteoglycan link protein 1 (HAPLN1) protein or the gene encoding it as an active ingredient.
- 청구항 1 내지 청구항 9 중 어느 한 항에 따른 조성물 및 청구항 10의 처리 지침을 포함하는 세포의 산화작용 예방 또는 억제용 키트.A kit for preventing or inhibiting oxidation of cells, comprising the composition according to any one of claims 1 to 9 and the processing instructions of claim 10.
- 청구항 1 내지 청구항 9 중 어느 한 항에 따른 조성물을 포함하는, 세포의 산화작용 예방 또는 억제와 관련된 실험용 시약 조성물.An experimental reagent composition related to preventing or inhibiting oxidation of cells, comprising the composition according to any one of claims 1 to 9.
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| HUYNH MAILEE, PAK CHOROM, MARKOVINA STEPHANIE, CALLANDER NATALIE S., CHNG KENNETH S., WUERZBERGER-DAVIS SHELLY M., BAKSHI DEBAYAN : "Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resistance in multiple myeloma", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 293, no. 7, 1 February 2018 (2018-02-01), US , pages 2452 - 2465, XP093135735, ISSN: 0021-9258, DOI: 10.1074/jbc.RA117.000667 * |
| KUŹNIK-TROCHA KORNELIA, WINSZ-SZCZOTKA KATARZYNA, LACHÓR-MOTYKA IWONA, DĄBKOWSKA KLAUDIA, WOJDAS MAGDALENA, OLCZYK KRYSTYNA, KOMOS: "The Effects of TNF-α Inhibition on the Metabolism of Cartilage: Relationship between KS, HA, HAPLN1 and ADAMTS4, ADAMTS5, TOS and TGF-β1 Plasma Concentrations in Patients with Juvenile Idiopathic Arthritis", JOURNAL OF CLINICAL MEDICINE, MULTIDISCIPLINARY DIGITAL PUBLISHING INSTITUTE (MDPI), CH, vol. 11, no. 7, 4 April 2022 (2022-04-04), CH , pages 2013, XP093135733, ISSN: 2077-0383, DOI: 10.3390/jcm11072013 * |
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