KR102340534B1 - Composition for preventing or treating rheumatoid arthritis comprising venom derived from Naja melanoleuca - Google Patents

Abstract

The present invention provides a pharmaceutical composition for preventing or treating rheumatoid arthritis, a quasi-drug composition, a food composition, a cosmetic composition, and the treatment of rheumatoid arthritis comprising snake venom derived from Naja melanoleuca, a kind of snake, an extract or a fraction thereof it's about how to Since the snake venom derived from Naja melanoleuca of the present invention can improve, alleviate or treat the symptoms of rheumatoid arthritis, a type of autoimmune disease, it will be widely used in the development of therapeutic agents for various autoimmune diseases including rheumatoid arthritis.

Description

Composition for preventing or treating rheumatoid arthritis comprising venom derived from Naja melanoleuca

The invention rheumatoid to that, the present invention more specifically relates to a baemdok composition for preventing or treating rheumatoid arthritis, including derived from Naja melanoleuca comprises a baemdok, its extracts or fractions thereof derived from melanoleuca a type of snake Naja It relates to a pharmaceutical composition for preventing or treating arthritis, a quasi-drug composition, a food composition, a cosmetic composition, and a method of treating rheumatoid arthritis using the composition.

In general, arthritis refers to a disease in which inflammation occurs in the damaged area after the joint is damaged, resulting in the joint area not recovering, and it is known that inflammation occurs in the damaged area and causes severe pain. . Although such arthritis is considered a type of inflammatory disease due to inflammation, which is the final symptom, inflammation is a kind of natural in vivo response accompanied by joint damage in vivo, and since the inflammation does not cause the onset of arthritis, it is generally Arthritis is classified into osteoarthritis, a type of degenerative disease, and rheumatoid arthritis, a type of immune disease, depending on the cause of joint damage.

Among them, osteoarthritis is defined as a chronic degenerative disease in which the articular cartilage is gradually damaged due to degeneration of the extracellular matrix constituting the articular cartilage and is accompanied by inflammation and pain, mainly in middle-aged or old age. Osteoarthritis prevalence is about 40% in those over 50 years of age, and as the age increases, the prevalence is higher in women than in men. The causes of osteoarthritis include degenerative changes, immune system abnormalities, infections, trauma, and metabolic disorders. Factors related to the onset of osteoarthritis include nitric oxide (NO), cytokines, and proteolytic enzymes. is known

In addition, rheumatoid arthritis is a type of representative systemic chronic autoimmune disease that mainly affects the joints, and it is known that genetic-environmental factors act in its pathogenesis, and genetic factors contribute about 60%. In rheumatoid arthritis, if disease activity is not controlled, irreversible joint deformation and disability occur over time, and it is known that joint destruction is affected by the degree of drug response to immunosuppressive agents and biological agents to control this.

As such, unlike osteoarthritis, which occurs naturally with increasing age, it is impossible to predict the onset time of rheumatoid arthritis, and since it is a type of innate autoimmune disease, it is impossible to cure it. A lot of research is being conducted in the direction of alleviating inflammation, and research is being actively conducted to develop anti-inflammatory agents mainly for the purpose of alleviating pain caused by inflammation. For example, non-steroidal anti-inflammatory drugs, a type of anti-inflammatory drug, have been developed for the purpose of alleviating the symptoms of rheumatoid arthritis. Therefore, the development of a more effective therapeutic agent is required.

On the other hand, snake venom refers to a mixture of various toxins secreted from poison glands, which are special salivary glands of poisonous snakes, and is known to include various proteases, including proteases, and various polypeptides that are toxic to cells. It is known that such snake venom is relatively strong against oxidation, and even if detoxified, its recovery is reversible, but it is very weak against reduction and is simply detoxified by cysteine. Recently, the pharmacological activity of the snake venom has been investigated, and accordingly, development as a therapeutic agent for treating various diseases is accelerating. For example,

Korean Patent Application Laid-Open No. 10-2009-0036908 discloses a composition for preventing or treating arteriosclerosis containing saxatilin, a disintegrin derived from snake venom, and Korean Patent Publication No. 10-2016-0118417 discloses a composition containing snake venom. A composition for alleviation or treatment of atopic skin disease is disclosed, and Japanese Patent Application Laid-Open No. 2010-075194 discloses a formulation for the treatment of inflammatory response-related diseases comprising a complex containing snake venom as an active ingredient, registered in Korea Patent No. 10-1802514 discloses a pharmaceutical composition for preventing or treating inflammatory diseases comprising snake venom derived from NM (Naja melanoleuca) as an active ingredient.

Under this background, the present inventors made intensive research efforts to develop a formulation capable of effectively treating rheumatoid arthritis. As a result, it was confirmed that snake venom derived from Naja melanoleuca can improve symptoms of rheumatoid arthritis, a type of autoimmune disease, and the present invention The invention was completed.

The main object of the present invention is to provide a pharmaceutical composition for preventing or treating rheumatoid arthritis, comprising snake venom derived from Naja melanoleuca, an extract thereof, or a fraction thereof.

Another object of the present invention is to provide a quasi-drug composition for preventing or improving rheumatoid arthritis, comprising snake venom derived from Naja melanoleuca, an extract thereof, or a fraction thereof.

Another object of the present invention is to provide a food composition for preventing or improving rheumatoid arthritis, comprising snake venom derived from Naja melanoleuca, an extract thereof, or a fraction thereof.

Another object of the present invention is to provide a cosmetic composition for preventing or improving rheumatoid arthritis, comprising snake venom derived from Naja melanoleuca, an extract thereof, or a fraction thereof.

Another object of the present invention is to provide a method for treating rheumatoid arthritis using the composition.

The present invention provides a pharmaceutical composition for preventing or treating rheumatoid arthritis, comprising snake venom derived from Naja melanoleuca, an extract thereof, or a fraction thereof.

As used herein, the term "extract" refers to a liquid component obtained by immersing a target substance in various solvents and then extracting it for a certain period of time at room temperature or in a heated state, and a product such as a solid obtained by removing the solvent from the liquid component. means In addition, in addition to the result, it can be comprehensively interpreted as including all of the dilutions of the results, their concentrates, their preparations, and their purified products. Accordingly, the snake venom extract derived from Naja melanoleuca provided in the present invention is an extract obtained by extracting snake venom derived from Naja melanoleuca , a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, a prepared product of the extract, or It can be interpreted to include extracts of all formulations that can be formed using the extract itself and the extract, such as purified products or mixtures thereof.

The method for obtaining the snake venom extract derived from Naja melanoleuca of the present invention is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include hot water extraction, ultrasonic extraction, filtration, and reflux extraction, and these may be performed alone or in combination of two or more methods.

In the present invention, the type of solvent used for the extraction is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water, alcohol, or a mixed solvent thereof, and these may be used alone or in combination of one or more, and specifically water may be used. When alcohol is used as the solvent, alcohol having 1 to 4 carbon atoms may be specifically used.

As used herein, the term "fraction" refers to a result obtained by performing fractionation in order to separate a specific component or a specific group of components from a mixture including various components.

The fractionation method for obtaining the fraction in the present invention is not particularly limited, and may be performed according to a method commonly used in the art. Non-limiting examples of the fractionation method include a solvent fractionation method performed by treating various solvents, an ultrafiltration fractionation method performed by passing an ultrafiltration membrane having a constant molecular weight cut-off value, various chromatography (size, charge, hydrophobicity) or a chromatographic fractionation method for performing separation according to affinity), and combinations thereof. Specifically, there may be mentioned a method of obtaining a fraction from the extract by treating an extract obtained by extracting snake venom derived from Naja melanoleuca of the present invention with a predetermined solvent.

In the present invention, the type of the fractionation solvent used to obtain the fraction is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractionation solvent include water, a polar solvent such as an alcohol having 1 to 4 carbon atoms; hexane (Hexan), ethyl acetate (Ethyl acetate), chloroform (Chloroform), a non-polar solvent such as dichloromethane (Dichloromethane); or a mixed solvent thereof. These may be used alone or in combination of one or more, but is not limited thereto.

In addition, the extract or fraction may be prepared and used in the form of a dry powder after extraction, but is not limited thereto.

As used herein, the term "rheumatoid arthritis" is an inflammatory autoimmune disease that simultaneously affects multiple joints. means disease.

The autoimmune disease refers to a disease in which an abnormality in immune function occurs and immune cells responsible for immune function attack organs or tissues of our body.

As used herein, the term “prevention” refers to any action of suppressing or delaying rheumatoid arthritis by administering the composition containing the snake venom derived from Naja melanoleuca.

As used herein, the term "treatment" refers to any action in which the symptoms of rheumatoid arthritis are improved or beneficially changed by administration of the composition containing the snake venom derived from Naja melanoleuca.

The pharmaceutical composition of the present invention may include 0.001 to 80, specifically 0.001 to 70, more specifically 0.001 to 60% by weight of snake venom derived from Naja melanoleuca based on the total weight of the composition, but is not limited thereto.

In addition, the pharmaceutical composition may further include a pharmaceutically acceptable carrier, excipient or diluent commonly used in the preparation of the pharmaceutical composition, and the carrier may include a non-naturally occurring carrier. have. The carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the pharmaceutical composition is prepared according to a conventional method for each tablet, pill, powder, granule, capsule, suspension, internal solution, emulsion, syrup, sterilized aqueous solution, non-aqueous solution, suspension, emulsion, freeze-dried formulation, transdermal It can be formulated and used in the form of absorbents, gels, lotions, ointments, creams, patches, cataplasmas, pastes, sprays, skin emulsions, skin suspensions, transdermally delivered patches, drug-containing bandages or suppositories. Specifically, in the case of formulation, it may be prepared using a diluent or excipient such as a filler, a weight agent, a binder, a wetting agent, a disintegrant, and a surfactant commonly used. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. It can be prepared by adding various excipients, for example, wetting agents, sweetening agents, fragrances, preservatives, and the like, in addition to liquids for oral use and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As the base of the suppository, Witepsol, macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, and the like can be used.

Another embodiment of the present invention provides a method for treating rheumatoid arthritis, comprising administering the pharmaceutical composition to a subject suspected of rheumatoid arthritis other than humans.

As used herein, the term “administration” refers to the act of introducing a composition containing snake venom derived from Naja melanoleuca to an individual by an appropriate method.

As used herein, the term “individual” refers to all animals, including humans, rats, mice, and livestock that have or can develop rheumatoid arthritis. As a specific example, it may be a mammal including a human.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the type and severity of the subject, age, sex, activity of the drug, Sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field. For example, the snake venom derived from Naja melanoleuca may be administered at a dose of 0.01 to 500 mg/kg per day, specifically 0.1 to 50 mg/kg, and the administration may be administered once or several times a day. may be

The pharmaceutical composition may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. and may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art.

In addition, the pharmaceutical composition may be administered orally or parenterally (eg, intravenously, subcutaneously, dermally, intraperitoneally or topically) according to a desired method, and the dosage may vary depending on the patient's condition and weight, disease Although it varies depending on the degree, drug form, administration route, and time of administration, it may be appropriately selected by those skilled in the art.

Another embodiment of the present invention provides a quasi-drug composition for preventing or improving rheumatoid arthritis, comprising snake venom derived from Naja melanoleuca or a fraction thereof.

In this case, the definition of the extract, the fraction, rheumatoid arthritis, prevention and improvement is the same as described above.

As used herein, the term "quasi-drug" refers to fibers, rubber products or the like, which are used for the purpose of treating, alleviating, treating or preventing diseases of humans or animals, have weak action on the human body or do not directly act on the human body, or non-machines and similar products, which are products that are used for sterilization, insecticide, and similar purposes to prevent infection It refers to items that are not instruments, machines or devices, and items used for the purpose of pharmacologically affecting the structure and function of humans or animals that are not instruments, machines, or devices, except for external preparations for skin and personal hygiene. Also includes supplies.

When the snake venom derived from Naja melanoleuca of the present invention is added to a quasi-drug composition for the purpose of preventing or improving rheumatoid arthritis, the snake venom derived from Naja melanoleuca can be added as it is or used together with other quasi-drug ingredients, and can be used in a conventional method can be used appropriately. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prophylactic, health or therapeutic treatment).

The quasi-drug composition is not particularly limited thereto, but includes personal hygiene products, external preparations for skin, disinfectant cleaner, shower foam, wet tissues, detergent soap, hand wash, mask or ointment. The external preparation for skin is not particularly limited thereto, and specifically, it may be prepared and used in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel, or gel. The personal care product is not particularly limited thereto, and specifically, it may be soap, wet tissue, tissue, shampoo, toothpaste, hair care product, air freshener gel, or cleaning gel.

Another embodiment of the present invention provides a food composition for preventing or improving rheumatoid arthritis, comprising snake venom derived from Naja melanoleuca, an extract thereof, or a fraction thereof.

In this case, the definition of the extract, the fraction, rheumatoid arthritis, prevention and improvement is the same as described above.

As used herein, the term “improvement” refers to any action that at least reduces a parameter related to a condition to be treated, for example, the severity of a symptom by administration of a composition comprising the extract.

As used herein, the term "food" refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages , vitamin complexes, health functional foods, and health foods, and includes all foods in a conventional sense.

Since the snake venom derived from Naja melanoleuca of the present invention, its extract or its fractions do not induce side effects in vivo even when administered orally, when ingested, a high level of prevention or improvement effect of rheumatoid arthritis can be expected, so health It can be very useful for promotion purposes.

The functional food (functional food) is the same term as food for special health use (FoSHU), and in addition to nutritional supply, it is processed to efficiently exhibit bioregulatory functions and has high medical effects. means food. Here, 'function (sex)' refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological effects on the structure and function of the human body. The food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, the formulation of the food can be prepared without limitation as long as it is a formulation recognized as a food.

The food composition of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage of not having side effects that may occur during long-term administration of drugs by using natural substances as raw materials, and has excellent portability, so the present invention Foods of rheumatoid arthritis can be consumed as a supplement to enhance the prevention or improvement effect.

The health food means food having an active health maintenance or promotion effect compared to general food, and health supplement food means food for the purpose of health supplementation. In some cases, the terms health functional food, health food, and dietary supplement are used interchangeably.

Specifically, the health functional food is prepared by adding snake venom derived from Naja melanoleuca of the present invention, an extract thereof, or a fraction thereof to food materials such as beverages, teas, spices, gum, confectionery, etc., or encapsulating, powdering, suspension, etc. As a food, it means that certain health effects are brought about when ingested, but unlike general drugs, it has the advantage that there are no side effects that may occur when taking the drug for a long time using food as a raw material.

The food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited and any carrier commonly used in the art may be used.

In addition, the food composition may include additional ingredients that are commonly used in food compositions to improve odor, taste, vision, and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr); and amino acids such as lysine, tryptophan, cysteine, and valine.

In addition, the food composition includes a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), a disinfectant (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), an antioxidant (butylhydroxyanisole (BHA), butyl hydro Loxytoluene (BHT), etc.), coloring agents (tar pigments, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasonings (MSG sodium glutamate, etc.), sweeteners (dulcin, cyclemate, saccharin, etc.) , sodium, etc.), flavorings (vanillin, lactones, etc.), swelling agents (alum, D-potassium hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickening agents (flavors), film agents, gum base agents, foam inhibitors, solvents, improving agents, etc. It may contain food additives. The additive may be selected according to the type of food and used in an appropriate amount.

As an example of the food composition of the present invention, it may be used as a health drink composition, and in this case, it may contain various flavoring agents or natural carbohydrates as an additional component like a conventional drink. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as taumatine, stevia extract; A synthetic sweetener such as saccharin or aspartame may be used. The ratio of the natural carbohydrate may be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per 100 ml of the health beverage composition of the present invention.

In addition to the above, the health drink composition includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid, pectic acid salts, alginic acid, alginic acid salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, It may contain alcohol, a carbonation agent, or the like. In addition, it may contain the pulp for the production of natural fruit juice, fruit juice beverage, or vegetable beverage. These components may be used independently or in combination. Although the ratio of these additives is not very important, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.

Another embodiment of the present invention provides a cosmetic composition for preventing or improving rheumatoid arthritis, comprising snake venom derived from Naja melanoleuca or a fraction thereof.

In this case, the definition of the extract, the fraction, rheumatoid arthritis, prevention and improvement is the same as described above.

The snake venom derived from Naja melanoleuca may include the snake venom derived from Naja melanoleuca in a proportion of 0.1% to 20% by weight based on the total weight of the composition, like the pharmaceutical composition. Specifically, it is included in a proportion of 0.1 wt% to 5 wt%, more specifically 0.4 to 0.6 wt%, and still more specifically 0.5 wt%, based on the total weight of the composition, but is not limited thereto.

Components included in the cosmetic composition of the present invention include components commonly used in cosmetic compositions in addition to snake venom derived from Naja melanoleuca , for example, water, surfactant, moisturizing agent, lower alcohol, chelating agent, bactericide, oxidation agent It may further include one or more additives selected from the group consisting of inhibitors, preservatives, colorants and fragrances.

In addition, the cosmetic composition may be prepared in any conventionally prepared formulation, for example, a solution, emulsion, suspension, paste, cream, lotion, gel, powder, spray, surfactant-containing cleansing agent, oil, soap, It may be formulated as a liquid detergent, bath agent, foundation, makeup base, essence, lotion, foam, pack, softening water, sunscreen cream or sun oil, but is not limited thereto.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Adult cellulose, aluminum metahydroxide, bentonite, or tracanth may be used.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. can

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additional chlorofluorohydrocarbon, propane /may contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide as carrier components Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester may be used.

In addition, the ingredients included in the cosmetic composition may be included in an amount generally used in the field of dermatology.

Since the snake venom derived from Naja melanoleuca of the present invention can improve, alleviate or treat the symptoms of rheumatoid arthritis, a type of autoimmune disease, it will be widely used in the development of therapeutic agents for various autoimmune diseases including rheumatoid arthritis.

FIG. 1 is a photograph of the damaged joints of arthritic mice treated derived baemdok samples from Naja melanoleuca, 2 derived baemdok from Naja melanoleuca the joints in arthritis week once / day symptoms after application to the skin external preparations It was confirmed that the group applied with PBS used as a control did not improve but worsened.
2 is a graph showing changes in the score of the recovered arthritis index of arthritis model mice treated with a snake venom sample derived from Naja melanoleuca and MTX (methotrexate).
3 is a graph showing the results of flow cytometry analysis of CCR1-positive cells targeting peripheral blood monocytes and lymph nodes of arthritis model mice orally administered MTX.
4 is a graph showing the results of flow cytometry analysis of CCR1-positive cells on peripheral blood monocytes and lymph nodes of arthritis model mice treated with snake venom samples derived from Naja melanoleuca.
Figure 5a is a graph showing the results of measuring the change in CD4 positive cells according to the dose in normal mice orally administered snake venom derived from Naja melanoleuca.
Figure 5b is a graph showing the results of measuring the change in CD25-positive cells according to the dose in normal mice orally administered snake venom derived from Naja melanoleuca.
Figure 5c is a graph showing the results of measuring the change in Foxp3-positive cells according to the dose in normal mice orally administered snake venom derived from Naja melanoleuca.
6A is a graph showing the results of measuring the changes in CD4 and CD25 positive cells according to the dose in normal mice orally administered snake venom derived from Naja melanoleuca.
Figure 6b is a graph showing the results of measuring the changes in CD25 and Foxp3-positive cells according to the dose in normal mice orally administered snake venom derived from Naja melanoleuca.
6c is a graph showing the results of measuring changes in CD4 and Foxp3-positive cells according to the dose in normal mice orally administered with snake venom derived from Naja melanoleuca.
6D is a graph showing the results of measuring changes in CD4, CD25 and Foxp3-positive regulatory T cells according to the dose in normal mice orally administered snake venom derived from Naja melanoleuca.
7a is a graph showing the results of comparison of CRP (C-reactive protein) levels in plasma in normal mice to which snake venom derived from Naja melanoleuca was orally administered or applied to the skin.
7B is a graph showing the results of comparing the level of aspartate aminotransferase (AST) in plasma in normal mice to which snake venom derived from Naja melanoleuca was orally administered or applied to the skin.
Figure 7c is a graph showing the results of comparing the level of ALT (alanine transaminase) in plasma in normal mice orally administered or skin-applied snake venom derived from Naja melanoleuca.
8 is a graph showing the results of comparing the level of IL-17 in plasma in normal mice orally administered snake venom derived from Naja melanoleuca.
9 is a photomicrograph showing the joint tissue of an arthritis model mouse that was orally administered with MTX or snake venom derived from Naja melanoleuca was applied to the skin.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Construction of Arthritis Model Mice

Type 2 collagen solution (10 mM), adjuvant (Complete freund's adjuvant) and oil (mineral oil) were mixed at a ratio of 1:1:1 (v/v/v) to obtain a mixture, and 50 μl of the mixture was added to 8 Injections were made into the tail vein 1 cm from the base of the tail of week-old ICR mice, and after 10 days, the injection was repeated once more. Mice exhibiting arthritis symptoms at 10 to 15 days after injection were selected and used as rheumatoid arthritis model mice.

On the other hand, in order to distinguish in detail the level of symptoms of the selected rheumatoid arthritis model mice, a severity score is given as follows (Table 1), and when the severity score is 2 or more, rheumatoid arthritis is was determined to be ill.

Injury Severity Score and Symptom Criteria in Arthritis Model Mice score Symptom 0 Normal condition with no symptoms One A condition that causes minimal swelling or redness in any one of the toes, insteps, or ankles 2 Significant swelling and redness in at least two of the toes, insteps, and ankles 3 Inflammation and swelling in three joints from the toes to the ankles 4 Significant swelling and swelling are observed all over the area from the toe to the ankle, or inability to use the foot freely due to stiffness

Meanwhile, the snake venom sample derived from Naja melanoleuca used in the present invention was purchased from Sigma-Aldrich and used.

Example 2: Symptoms of Arthritis Model Mice Naja melanoleuca The therapeutic effect of snake venom derived from

A snake venom sample derived from Naja melanoleuca was applied to the skin of the damaged joint area of the arthritis model mouse prepared in Example 1 for 2 weeks, and the therapeutic effect of the snake venom was visually verified ( FIG. 1 ). At this time, as a control, mice coated with PBS were used.

1 is a photograph showing the damaged joint area of an arthritis model mouse treated with a snake venom sample derived from Naja melanoleuca.

In addition, a snake venom sample derived from Naja melanoleuca was treated in the joint area of the four injured limbs (one pair of forelimbs and one pair of hind limbs) of an arthritis model mouse, and then each injury severity score was measured ( FIG. 2 ). In this case, as a control group, PBS-coated mice were used, and as a control group, mice orally administered with MTX (methotrexate), known as a treatment for rheumatoid arthritis, were used.

2 is a graph showing the changes in the recovered arthritis index score of arthritis model mice treated with a snake venom sample derived from Naja melanoleuca and MTX (methotrexate), D1 represents the time point 1 day after treatment, and D11 is after treatment 11 days have elapsed, (○) represents each arthritis model mouse, (-) is the injury severity score measured at 1 day in the same arthritis model mouse, and 11 days in the same arthritis model mouse. Changes in the measured damage severity score are linked, and red marks indicate the average value of the damage severity score measured at 1 day and 11 days, respectively.

As shown in Figure 2, in the case of the control group, the damage severity score measured on days 1 and 11 did not show a significant difference, but when MTX was orally administered (comparative group) or treated with a snake venom sample derived from Naja melanoleuca, It was confirmed that the damage severity score measured on the 11th day was significantly reduced compared to the damage severity score measured on the 1st day.

Therefore, it was found that the snake venom sample derived from Naja melanoleuca showed the effect of improving the symptoms of rheumatoid arthritis.

Example 3: Flow cytometry analysis in arthritis model mice

Example 3-1: Changes in CCR1-positive cells in arthritis model mice orally administered methotrexate

Peripheral blood mononuclear cells (PBMC) and lymph nodes (LN) were obtained from arthritis model mice orally administered with MTX, and flow cytometry was performed on them.

Roughly, an anti-mouse CCR1 antibody was added to peripheral blood mononuclear cells and cells isolated from lymph nodes, and reacted at 4°C for 30 minutes. Then, in order to perform flow cytometry, a BD cantoII flow cytometer was used (FIG. 3). At this time, as a control, mice administered with PBS were used.

3 is a graph showing the results of flow cytometry analysis of CCR1-positive cells targeting peripheral blood monocytes and lymph nodes of an arthritis model mouse orally administered with MTX (methotrexate).

As shown in FIG. 3 , it was found that the level of CCR1-positive cells in the cell surface and cytoplasm of peripheral blood monocytes and lymph nodes was increased in arthritis model mice orally administered with MTX, known as a therapeutic agent for rheumatoid arthritis.

From the result of increasing the level of the CCR1-positive cells, it was found that the symptoms of inflammation were improved.

Example 3-2: Naja melanoleuca Changes in CCR1-positive cells in arthritis model mice treated with snake venom derived from

A snake venom sample derived from Naja melanoleuca was applied to the skin of the damaged joint area of the arthritis model mouse prepared in Example 1 for 2 weeks, and peripheral blood mononuclear cells (PBMC) and lymph nodes (LN) were obtained from the mouse. After obtaining it, it was subjected to flow cytometry by the method of Example 3-1 (FIG. 4). At this time, as a control, mice coated with PBS were used.

4 is a graph showing the results of flow cytometry analysis of CCR1-positive cells on peripheral blood monocytes and lymph nodes of arthritis model mice treated with snake venom samples derived from Naja melanoleuca.

As shown in FIG. 4 , it was confirmed that the level of CCR1-positive cells in peripheral blood mononuclear cells and lymph nodes was increased in arthritis model mice treated with snake venom of the present invention.

These results are similar to the results of oral administration of MTX, which is known as a therapeutic agent for rheumatoid arthritis, shown in FIG. 3, and compared with the results of FIG. 3, it was found that the snake venom of the present invention exhibits a therapeutic effect on rheumatoid arthritis.

Example 4: Flow cytometry analysis in normal mice

Example 4-1: Naja melanoleuca Changes in CD4, CD25 and Foxp3-positive cells in normal mice orally administered snake venom derived from

The purpose of this study was to determine whether snake venom derived from Naja melanoleuca could increase the frequency of regulatory T cells.

To this end, 0.1 μg or 1 μg of a snake venom sample derived from Naja melanoleuca was orally administered to normal mice, and the levels of CD4-positive cells, CD25-positive cells and Foxp3-positive cells were confirmed through flow cytometry (FIG. 5a to 5c). At this time, as a control group, mice orally administered PBS were used. In addition, CD4-positive cells and CD25-positive cells were confirmed by the method of Example 3-1. Finally, Foxp3-positive cells were confirmed by intranuclear staining. Roughly, the cells to be stained were washed twice with 1x permeabilization buffer, then fixed with Fix/perm buffer, anti-mouse Foxp3 antibody was added, and then at 4°C. The reaction was carried out for 30 minutes.

Figure 5a is a graph illustrating results of measuring changes in CD4 positive cells in accordance with the derived baemdok from Naja melanoleuca the dose in oral administration of a normal mouse, Figure 5b derived baemdok from Naja melanoleuca in oral administration a normal mouse It is a graph showing the result of measuring the change in CD25-positive cells according to the dose, and FIG. 5c is a graph showing the result of measuring the change in the Foxp3-positive cells according to the dose in normal mice orally administered snake venom derived from Naja melanoleuca. to be.

As shown in FIGS. 5A to 5C , among CD4 positive, CD25 positive and Foxp3 positive cells known as regulatory T cell markers, the frequency of CD25 positive and Foxp3 positive cells was confirmed to be increased by oral administration of a snake venom sample derived from Naja melanoleuca. did

Thus, oral administration of the baemdok sample derived from Naja melanoleuca are regulatory T cells the main characteristics of CD25-positive and Foxp3 therefore let the frequency of positive cells increased individually, from which the oral administration of the baemdok sample derived from melanoleuca Naja control of T was analyzed to increase the level of cells.

Example 4-2: Naja melanoleuca Changes in regulatory T cells in normal mice orally administered snake venom derived from

Results of Example 4-1, since the oral administration of the baemdok sample derived from Naja melanoleuca provide clues will increase the levels of regulatory T cells, in fact, the oral administration of the baemdok sample derived from Naja melanoleuca regulatory T cells The purpose of this study was to determine whether it was possible to increase the level of

For this purpose, 0.1 μg or 1 μg of a snake venom sample derived from Naja melanoleuca was orally administered to normal mice, and the level of cells including CD4 positive, CD25 positive, and Foxp3 positive cells was confirmed through flow cytometry ( 6a to 6d). At this time, as a control group, mice orally administered PBS were used.

Figure 6a is a graph showing the results of measuring the changes in CD4 and CD25 positive cells according to the dose in normal mice orally administered snake venom derived from Naja melanoleuca, Figure 6b is a normal mouse orally administered snake venom derived from Naja melanoleuca It is a graph showing the results of measuring changes in CD25 and Foxp3-positive cells according to the dose in mice, and FIG. 6c is a graph showing the changes in CD4 and Foxp3-positive cells according to the dose in normal mice orally administered snake venom derived from Naja melanoleuca. It is a graph showing the measurement results, and FIG. 6d is a graph showing the results of measuring changes in CD4, CD25 and Foxp3-positive regulatory T cells according to the dose in normal mice orally administered snake venom derived from Naja melanoleuca. It is a graph.

As shown in FIGS. 6A to 6D , it was confirmed that the snake venom sample derived from Naja melanoleuca increased the levels of each combination of CD4 positivity, CD25 positivity and Foxp3 positivity, which are known major characteristics of regulatory T cells.

Therefore, it was found that oral administration of a snake venom sample derived from Naja melanoleuca can increase the level of regulatory T cells and, due to the increase in the level of these regulatory T cells, can improve the inflammatory symptoms of rheumatoid arthritis.

Example 5: Effect on metabolite levels in normal mice Naja melanoleuca The effect of snake venom derived from

Example 5-1: Blood level of C-reactive protein (CRP)

After oral administration or skin application of a snake venom sample derived from Naja melanoleuca to normal mice, plasma from each mouse was collected, and CRP (C-reactive protein) levels in plasma, an inflammatory marker, were compared ( FIG. 7a ). In this case, as a control group, mice to which PBS was orally administered or skin applied were used.

7a is a graph showing the results of comparison of CRP (C-reactive protein) levels in plasma in normal mice to which snake venom derived from Naja melanoleuca was orally administered or applied to the skin.

As shown in FIG. 7a , it was confirmed that the CRP level in plasma was lowered when the snake venom derived from Naja melanoleuca was administered orally or applied to the skin.

Thus, since the inflammatory markers in plasma CRP levels decreased by check baemdok derived from Naja melanoleuca, could poison derived from snake Naja melanoleuca know that you can improve the inflammation of rheumatoid arthritis.

Example 5-2: Blood levels of aspartate aminotransferase (AST)

After oral administration or skin application of a snake venom sample derived from Naja melanoleuca to normal mice, plasma from each mouse was collected, and AST (aspartate aminotransferase) levels in plasma were compared (FIG. 7b). In this case, as a control group, mice to which PBS was orally administered or skin applied were used.

7B is a graph showing the results of comparing the level of aspartate aminotransferase (AST) in plasma in normal mice to which snake venom derived from Naja melanoleuca was orally administered or applied to the skin.

As shown in FIG. 7b , when the snake venom derived from Naja melanoleuca was orally administered, there was no significant difference from the control group, but when applied to the skin, the plasma AST level showed a tendency to slightly increase.

Thus, because you did not even plasma AST levels by administering the oral baemdok derived from Naja melanoleuca changes, the baemdok derived from Naja melanoleuca was analyzed that would represent a much toxicity in the body, it is administered orally.

Example 5-3: Blood level of ALT (alanine transaminase)

After oral administration or skin application of a snake venom sample derived from Naja melanoleuca to normal mice, plasma was collected from each mouse, and the level of ALT (alanine transaminase) in plasma was compared ( FIG. 7c ). In this case, as a control group, mice to which PBS was orally administered or skin applied were used.

Figure 7c is a graph showing the results of comparing the level of ALT (alanine transaminase) in plasma in normal mice orally administered or skin-applied snake venom derived from Naja melanoleuca.

As shown in FIG. 7c , it was confirmed that when the snake venom derived from Naja melanoleuca was administered orally or applied to the skin, the plasma ALT level was lowered compared to the control group. In particular, it was confirmed that the plasma ALT level was relatively lower in the case of skin application than in the case of oral administration.

Thus, once again check if administered orally derived baemdok from Naja melanoleuca, therefore showed a tendency for the plasma ALT levels rather reduced, the baemdok derived from Naja melanoleuca is not shown in the little toxicity in the body, it is administered orally could

Example 6: Cytokine Level Analysis

After oral administration of a snake venom sample derived from Naja melanoleuca to normal mice, plasma from the mice was collected, and the level of IL-17 in the plasma was compared ( FIG. 8 ). In this case, as a control group, mice to which PBS was orally administered or skin applied were used.

8 is a graph showing the results of comparing the level of IL-17 in plasma in normal mice orally administered snake venom derived from Naja melanoleuca.

As shown in FIG. 8 , when the snake venom derived from Naja melanoleuca was orally administered, it was confirmed that the plasma IL-17 level was lowered compared to the control group.

That since the IL-17 is known as the inflammatory cytokines, resulting sikindaneun venom derived snake from the Naja melanoleuca decrease the IL-17 level, even be poisonous derived snake from Naja melanoleuca improve inflammatory symptoms of rheumatoid arthritis analyzed to mean

Example 7: Histological Analysis of Arthritis Model Mice

MTX was orally administered to the arthritis model mouse prepared in Example 1 or snake venom derived from Naja melanoleuca was applied to the skin, and arthritic tissue was collected from the mouse, a tissue slice thereof was obtained, and inflammation in the tissue In order to confirm the presence of cells, it was confirmed with a microscope (FIG. 9). At this time, as a control, mice orally administered PBS were used. In addition, the tissue slices were prepared by decalcifying the collected tissue in a decalcification solution, fixing it to make a paraffin block, cutting it to obtain a slice, and then performing H&E staining.

9 is a photomicrograph showing the joint tissue of an arthritis model mouse that was orally administered with MTX or snake venom derived from Naja melanoleuca was applied to the skin.

As shown in FIG. 9 , it was confirmed that inflammatory cells were present in the joints in the tissues of the control group, but in the tissues of arthritis model mice that were orally administered with MTX or snake venom derived from Naja melanoleuca was applied to the skin, inflammatory cells in the joints was confirmed not to exist.

Therefore, it was found that snake venom derived from Naja melanoleuca can improve the inflammatory symptoms of rheumatoid arthritis.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims to be described later and their equivalents.

Claims (8)

A pharmaceutical composition for preventing or treating rheumatoid arthritis, comprising a snake venom derived from Naja melanoleuca, an extract or a fraction thereof.
According to claim 1,
The extract is a pharmaceutical composition for preventing or treating rheumatoid arthritis, which is obtained by extracting snake venom derived from Naja melanoleuca with one or more solvents selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof.
According to claim 1,
The fraction is obtained by fractionating the snake venom extract with a solvent selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, hexane, ethyl acetate, chloroform, dichloromethane, and mixed solvents thereof. Prevention of rheumatoid arthritis or A therapeutic pharmaceutical composition.
According to claim 1,
The composition further comprises a pharmaceutically acceptable carrier, excipient or diluent, a pharmaceutical composition for the prevention or treatment of rheumatoid arthritis.
A method for treating rheumatoid arthritis, comprising administering the pharmaceutical composition of any one of claims 1 to 4 to a subject suspected of rheumatoid arthritis, excluding humans.
A quasi-drug composition for preventing or improving rheumatoid arthritis, comprising snake venom derived from Naja melanoleuca, an extract or a fraction thereof.
delete A cosmetic composition for preventing or improving rheumatoid arthritis, comprising snake venom derived from Naja melanoleuca, an extract thereof, or a fraction thereof.

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