JP5828018B2 - Rheumatoid arthritis therapeutic agent and pharmaceuticals containing the same - Google Patents

Description

  The present invention relates to a therapeutic agent for rheumatoid arthritis and a pharmaceutical agent containing the same.

  In recent years, an increasing number of patients suffer from inflammatory diseases due to rapid changes in eating habits and lifestyle habits. An example of an inflammatory disease is an autoimmune disease. An autoimmune disease is an immune system that is originally responsible for recognizing and eliminating foreign substances, such as bacteria, viruses, and tumors, and reacts excessively to its own normal cells and tissues. Is a general term for diseases that cause symptoms, and this is caused by abnormalities in immunocompetent cells such as T cells, B cells, and macrophages.

  In addition, since many autoimmune disease symptoms are chronic and are refractory, there are many diseases included in specific diseases that are set as public expenditure targets in Japan. Although the treatment method varies depending on the disease, steroids and immunosuppressive agents are used as the first-line drugs in many diseases because immune disorders are the cause of the disease. However, these drugs are known to have many side effects, and there is a demand for drugs for improving immune abnormalities with few side effects.

  Patent Document 1 describes that high molecular hyaluronic acid suppresses the expression of interleukin 12 that promotes inflammation. Interleukin 12 is one of the cytokines that promote inflammation in autoimmune diseases. However, since autoimmune diseases develop various symptoms at various sites, it is difficult to comprehensively improve immune diseases even if only a single cytokine is controlled.

  Moreover, arthritis is mentioned as another example of an inflammatory disease. Among arthritis, autoimmune arthritis is one of the autoimmune diseases, and against this autoimmune arthritis, Suppressor of cytokine signaling 3 (SOCS3) that regulates signal transduction through cytokines is an autoimmune arthritis. It has been reported that the bone destruction seen is significantly reduced (Non-patent Document 1). That is, if the expression of SOCS3 is promoted, inhibition of cytokine signaling occurs, suggesting the possibility of comprehensively improving autoimmune arthritis. Furthermore, SOCS3 is said to be strongly expressed in chronic inflammatory diseases such as human colitis and arthritis (Non-patent Document 2).

  In addition, SOCS3 has been reported to indirectly control Toll-like receptors in innate immune cells (Non-patent Document 3).

  Therefore, if it is possible to promote the expression of SOCS3, it is considered useful for the treatment and prevention of inflammatory diseases, but studies on the regulation of the expression of SOCS3 in inflammatory diseases have hardly been made so far.

WO2002-074318

Takanori Shouda, Takafumi Yoshida, Toshikatsu Hanada, Toru Wakioka, Masanobu Oishi, Kanta Miyoshi, Setsuro Komiya, Ken-ichiro Kosai, Yasushi Hanakawa, Koji Hashimoto, Kensei Nagata, and Akihiko Yoshimura, “Induction of the cytokine signal regulator SOCS3 / CIS3 as a therapeutic strategy for treating inflammatory arthritis ”, J. Clin. Invest., Vol. 108, No. 12, 1781-1788, December 2001 Hiromi Takagi, Takato Sanada, Yasumasa Hamada, Akihiko Yoshimura, “Control of Cytokines and Toll-like Receptor Signals by SOCS”, Linyi Nihon, Vol. 62, No. 12 (2004-12), 2189-2196 Andrea Baetz, Markus Frey, Klaus Heeg, and Alexander H. Dalpke, “Suppressor of Cytokine Signaling (SOCS) Proteins Indirectly Regulate Toll-like Receptor Signaling in Innate Immune Cells”, J. Biol. Chem., Vol. 279, Issue 52 , 54708-54715, December 24, 2004.

  Therefore, the present invention provides a therapeutic agent for rheumatoid arthritis that can comprehensively improve an inflammatory disease (rheumatoid arthritis) and a medicine containing the same.

  As a result of intensive studies on the expression of SOCS3, the present inventors have unexpectedly found that the expression of SOCS3 is promoted by ingesting hyaluronic acid and / or a salt thereof having an average molecular weight of 500,000 or more. The present invention has been completed.

  The therapeutic agent for rheumatoid arthritis according to one embodiment of the present invention contains hyaluronic acid having an average molecular weight of 500,000 or more and / or a salt thereof as an active ingredient.

  In the above rheumatoid arthritis therapeutic agent, the expression of SOCS3 is promoted by ingestion by humans or animals other than humans. As a result, the symptoms of rheumatoid arthritis can be alleviated.

  In the rheumatoid arthritis therapeutic agent, an average particle size of the hyaluronic acid and / or a salt thereof may be 50 to 500 μm.

  The pharmaceutical agent which concerns on 1 aspect of this invention contains the said rheumatoid arthritis therapeutic agent as an active ingredient. In this case, 10 to 1000 mg of the medicine can be taken per day.

  According to the above rheumatoid arthritis therapeutic agent, the expression of SOCS3 is promoted by ingesting hyaluronic acid and / or a salt thereof having an average molecular weight of 500,000 or more, and the symptoms of inflammatory diseases (rheumatoid arthritis) are comprehensively improved. It becomes possible.

  Therefore, it is possible to provide a pharmaceutical agent for improving inflammatory diseases that has no or very little side effect, and can be expected to further expand the use of hyaluronic acid and / or a salt thereof.

1 is a photograph of electrophoresis in which SOCS3 gene expression by RT-PCR was examined in Example 1. FIG. From the left, the results of the distilled water intake group, the prototype A intake group, and the prototype B intake group are shown. FIG. 2 is a photograph of the surface of the intestinal epithelium of a mouse orally ingested with hyaluronic acid or a salt thereof in Example 1. FIG. 3 is a photograph of the surface of the large intestine epithelium of a mouse orally ingested with hyaluronic acid or a salt thereof in Example 1. FIG. 4 is a photograph of the surface of the large intestine epithelium (after double staining treatment) of a mouse orally ingested with hyaluronic acid or a salt thereof in Example 1. FIG. 5 is a photograph of a cervical lymph node of a mouse administered with drinking water of distilled water or a hyaluronic acid prototype in Example 1. FIG. 6 is a photograph of electrophoresis in which SOCS3 gene expression by RT-PCR was examined in Example 2. From the left, the results are shown with no hyaluronic acid added, prototype A added, and prototype B added.

  Hereinafter, a SOCS3 expression promoter according to an embodiment of the present invention, a pharmaceutical and a food containing the same, a method for promoting the expression of SOCS3, a proiotrophin expression inhibitor, a pharmaceutical and a food containing the same, and A method for suppressing the expression of proiotrophin will be described in detail.

  In the present invention, “%” means “mass%”.

1. SOCS3 expression promoter, pharmaceutical and food containing the same, and method for promoting SOCS3 expression 1.1. SOCS3 Expression Promoter The SOCS3 expression promoter according to one embodiment of the present invention is characterized by containing hyaluronic acid having an average molecular weight of 500,000 or more and / or a salt thereof as an active ingredient.

  Here, “hyaluronic acid” refers to a polysaccharide having one or more repeating structural units composed of disaccharides of glucuronic acid and N-acetylglucosamine. The “hyaluronic acid salt” is not particularly limited, but is preferably a food or pharmaceutically acceptable salt, for example, sodium salt, potassium salt, calcium salt, zinc salt, magnesium salt, ammonium salt, etc. Is mentioned.

  In addition, the average molecular weight of hyaluronic acid and / or a salt thereof used in the SOCS3 expression promoter according to this embodiment is 500,000 or more, preferably 600,000 or more, more preferably 500,000 to 1.6 million, and still more preferably 60. 10 to 1.6 million. When the average molecular weight of hyaluronic acid and / or a salt thereof is less than 500,000, it is difficult to improve the symptoms of inflammatory diseases and the function of the immune system may be reduced. Moreover, when the average molecular weight of hyaluronic acid and / or a salt thereof exceeds 1.6 million, it may be difficult to dissolve and the effect thereof may not be sufficiently exerted, or it may be difficult to add to foods.

  The measurement method of the average molecular weight prescribed | regulated by this invention is demonstrated.

  That is, about 0.05 g of purified hyaluronic acid was accurately weighed and dissolved in a 0.2 mol / L sodium chloride solution to make exactly 100 mL, and 8 mL, 12 mL and 16 mL of this solution were accurately weighed, and 0 A solution obtained by adding a 2 mol / L sodium chloride solution to make exactly 20 mL is used as a sample solution. This sample solution and a 0.2 mol / L sodium chloride solution were 30.0 ± 0.1 ° C. according to the viscosity measurement method (first method capillary viscometry method) of the Japanese Pharmacopoeia (14th revision) general test method. The specific viscosity is measured by (Equation (1)), and the reduced viscosity at each concentration is calculated (Equation (2)). A graph is drawn with the reduced viscosity on the vertical axis and the concentration (g / 100 mL) of the product converted to dry matter on the horizontal axis, and the intrinsic viscosity is determined from the intersection of the straight line connecting the points and the vertical axis. Substituting the intrinsic viscosity obtained here into Laurent's formula (Formula (3)) to calculate the average molecular weight (TC Laurent, M. Ryan, A. Pietruszkiewicz,: BBA, 42, 476-485 (1960)) .

(Formula 1)
Specific viscosity = {(required flow time for sample solution) / (required flow time for 0.2 mol / L sodium chloride solution)}-1

(Formula 2)
Reduced viscosity = specific viscosity / (concentration of the product in terms of dry matter (g / 100mL))

(Formula 3)
Intrinsic viscosity = 3.6 × 10 ⁻⁴ M ^0.78
M: average molecular weight

  The hyaluronic acid contained in the SOCS3 expression promoter according to this embodiment is basically a disaccharide unit in which the 1-position of β-D-glucuronic acid and the 3-position of β-DN-acetyl-glucosamine are combined. Having at least one disaccharide and basically composed of β-D-glucuronic acid and β-DN-acetyl-glucosamine, wherein a plurality of disaccharide units are combined, Alternatively, it may be a sugar to which these elements are bonded, and derivatives thereof such as those having a hydrolyzable protecting group such as an acyl group can also be used. The sugar may be an unsaturated sugar, and examples of the unsaturated sugar include non-reducing terminal sugars, usually those having unsaturated carbon atoms between positions 4 and 5 of glucuronic acid.

  Hyaluronic acid and / or a salt thereof may be extracted from natural products such as animals (for example, biological tissues such as chicken crown, umbilical cord, skin, joint fluid, etc.), or obtained by culturing microorganisms or animal cells. And the like (for example, fermentation using a bacterium belonging to the genus Streptococcus, etc.), or those synthesized chemically or enzymatically can be used.

  As the hyaluronic acid used in the SOCS3 expression promoter according to the present embodiment, a commercially available product can be used. For example, it can also be produced according to the following production methods 1 and 2.

1.1.1. Production method 1 (extraction from chicken crown)
First, the chicken crown is heat-treated. This is because the protein contained in the chicken crown is heat denatured or the enzyme is deactivated. Any method may be used for the heat treatment, but the heat treatment can be efficiently performed by immersing the chicken crown in hot water. The heating temperature and time are not particularly limited as long as the protein in the chicken crown is denatured or the enzyme is deactivated. When the heating method using hot water is adopted, the heat of 60 to 100 ° C. The raw material is preferably immersed in water for 20 to 90 minutes.

  In addition, when using a frozen chicken crown, the chicken crown may be heated as it is, but it is preferable to heat the frozen chicken crown after putting it in running water or the like and then subjecting it to a heat treatment so that a certain quality can be obtained.

  Next, the heat-treated chicken crown is made into a paste. This pasting improves the yield of hyaluronic acid. Prior to pasting, if the chicken crown after the heat treatment is cut into thin pieces with a shredder or shredded with a chopper for grinding meat, it becomes easy to paste. An example of pasting is to add approximately 1 to 5 times the amount of fresh water to the chicken crown, and homogenize with a homogenizer for 10 to 60 minutes, so that the chicken crown is crushed and made into fine particles and can be finished into a paste. . In addition, you may use a high-speed stirrer and a crusher other than a homogenizer for pasting.

  Next, acid agents such as hydrochloric acid and sulfuric acid, or alkali agents such as sodium hydroxide and potassium hydroxide are added to pasted chicken crowns to reduce the molecular weight of hyaluronic acid by acid treatment or alkali treatment. The average molecular weight of the acid is adjusted to 500,000 or more. As a preparation method, the concentration of the acid agent or the alkali agent, the addition amount and the treatment time may be appropriately combined so that the treated hyaluronic acid has a desired molecular weight. It is preferable because the molecular weight can be easily controlled. As an example of alkali treatment, after adding about 1 to 5% alkaline aqueous solution with a concentration of 10 to 30% to the chicken crown and treating at 25 to 70 ° C. for about 15 to 90 minutes, Neutralize with hydrochloric acid to adjust the molecular weight.

  Next, a protease is added to the raw material whose molecular weight has been adjusted, and protease treatment is performed. Any proteolytic enzyme can be used as long as it is commercially available, and examples thereof include pepsin, trypsin, papain, promeline and the like. The amount of the protease added is suitably 0.01 to 1% with respect to the chicken crown. In addition, the temperature and time of protease treatment are suitably in the range of 35 to 65 ° C. and 1 to 10 hours.

  Finally, hyaluronic acid is fractionated from the obtained protease-treated product to obtain crude hyaluronic acid, and then the hyaluronic acid is purified to obtain hyaluronic acid having a purity of 90% or more and an average molecular weight of 500,000 or more. It is done.

  Here, the fractionation and purification of hyaluronic acid can be performed according to a conventional method. For example, first, the protease-treated raw material is filtered to remove solids to obtain a filtrate containing crude hyaluronic acid. Prior to filtration, activated carbon may be added to the protease-treated product for the purpose of deodorization / decolorization or removal of some protein degradation products. And after melt | dissolving salt in the obtained filtrate, ethanol is added and hyaluronic acid is precipitated, and a deposit is fractionated. Thereafter, hydrous ethanol having an ethanol concentration of about 80 to 95% by volume is added to the precipitate, washed with a homogenizer, and the precipitate is collected. The hyaluronic acid of Production Example 1 can be obtained by repeating this washing with hydrous ethanol about 2 to 10 times and drying the collected precipitate.

1.1.2. Production method 2 (microbe fermentation method)
Hyaluronic acid calculation Activated carbon is added to the culture solution of Streptococcus microorganisms (Streptococcus zoopidemicus), followed by deodorization and decolorization, followed by filtration. Sodium chloride is dissolved in the obtained filtrate, ethanol is added to precipitate hyaluronic acid, and the precipitate is collected. Thereafter, hydrous ethanol having an ethanol concentration of about 80 to 95% by volume is added to the precipitate, washed with a homogenizer, and the precipitate is collected. The hyaluronic acid (average molecular weight of 500,000 or more) of Production Example 2 can be obtained by repeating this washing with water-containing ethanol about 2 to 10 times and drying the collected precipitate.

  In addition, the purity of the hyaluronic acid used in the present invention may be a level that can be used in pharmaceuticals or foods, preferably 90% or more, and more preferably 95% or more. This purity is defined as a value obtained by removing impurities other than hyaluronic acid from 100% in terms of dry matter. Here, examples of the impurities include protein degradation products, fat (crude fat), chondroitin sulfate, and the like. Specifically, the purity of hyaluronic acid using chicken crown as a raw material can be obtained by the following formula (4).

(Formula 4)
Hyaluronic acid purity (%) = 100-proteolysate (%)-crude fat (%)-chondroitin sulfate (%)

  In Formula 4, the protein degradation product (%) is a value obtained by the Lowry method, and the crude fat (%) is a new food analysis method (published by Korin Co., Ltd.) “Chapter 1 General components and related components, 1- “4 lipids, 1-4-2 ether extraction method”, and chondroitin sulfate (%) is a value obtained by the method described below.

  First, hyaluronic acid is dried, 50 mg of it is accurately weighed, and purified water is added to dissolve it. Make exactly 100 mL to make a test solution, take 4 mL of the test solution in a test tube, and add 1 mL of 0.5 mol / L sulfuric acid. Then, 0.2 mL of 0.04 mol / L cetyltrimethylammonium bromide was added to the solution obtained by heating in a water bath for 10 minutes and then cooling, and the mixture was allowed to stand at room temperature for 1 hour. Absorbance at a length of 10 mm and a wavelength of 660 nm is measured.

  Next, the obtained absorbance data is applied to a calibration curve for chondroitin sulfate to determine the amount (%) of chondroitin sulfate in purified hyaluronic acid. Here, the calibration curve is obtained by drying (depressurization, phosphorus pentoxide, 60 ° C., 5 hours) of chondroitin sulfate A sodium salt (SG (Special Grade), manufactured by Seikagaku Corporation) derived from whale cartilage. Precisely weigh and dissolve in purified water, and prepare solutions containing 10 μg, 20 μg, 30 μg, and 40 μg of chondroitin sulfate A sodium salt in 1 mL, respectively. For each 4 ml of solution, 1 mL of 0.5 mol / L sulfuric acid Then, 0.2 mL of 0.04 mol / L cetyltrimethylammonium bromide was added and mixed. After standing at room temperature for 1 hour, the absorbance was measured in the same manner. It was prepared by plotting the corresponding chondroitin sulfate A sodium salt solution (μg / mL) on the horizontal axis.

  The promotion of SOCS3 expression in cells or tissues of human or non-human animals can be achieved by, for example, Northern blotting, detection or quantification of SOCS3 gene using a DNA array, DNA chip, etc., and Western blotting, ELISA, affinity chromatography, etc. It can be confirmed by a known biochemical analysis method such as detection or quantification of SOCS3.

  The SOCS3 expression promoter according to one embodiment of the present invention usually contains 0.1% by mass or more of hyaluronic acid and / or a salt thereof having an average molecular weight of 500,000 or more, preferably 0.5 to 100% by mass. contains.

  Moreover, it is preferable that the average particle diameter of hyaluronic acid and / or its salt is 50-500 micrometers, It is more preferable that it is 80-500 micrometers, It is further more preferable that it is 200-500 micrometers.

  The average particle size of hyaluronic acid and / or its salt (hereinafter referred to as “hyaluronic acid” in this paragraph) having an average molecular weight of 500,000 or more (preferably 600,000 or more, more preferably 600,000 to 1.6 million) is 50. Since the powdered hyaluronic acid and / or its salt taken orally ingested in the gastric juice is dissolved at an appropriate rate by being ~ 500 μm, the hydrolysis of hyaluronic acid in the gastric juice is suppressed, Hyaluronic acid can be absorbed from the intestinal tract while maintaining the molecular weight and can reach the affected area. Here, when the average particle size of hyaluronic acid is less than 50 μm, hyaluronic acid dissolves rapidly in the gastric juice and hydrolysis proceeds in the gastric juice, resulting in a decrease in molecular weight. As described above, hyaluronic acid having a reduced molecular weight (for example, a molecular weight of 500,000 or less) is difficult to improve the symptoms of inflammatory diseases and may reduce the function of the immune system. On the other hand, when the average particle size of hyaluronic acid exceeds 500 μm, hyaluronic acid may not be completely dissolved in the body when ingested, and the amount absorbed may be less than the ingested amount. As a result, a sufficient effect for improving the symptoms of the inflammatory disease may not be obtained.

  In the present invention, the average particle size of hyaluronic acid and / or a salt thereof is measured by a laser diffraction scattering method. The average particle size of hyaluronic acid and / or a salt thereof can be adjusted by a commonly used method such as pulverization or sieving.

1.2. Pharmaceutical Product The pharmaceutical product according to one embodiment of the present invention contains the SOCS3 expression promoter according to the present embodiment as an active ingredient. More specifically, the pharmaceutical product according to the present embodiment can contain 5 to 100% by mass of the SOCS3 expression promoter according to the present embodiment.

  The pharmaceutical product according to the present embodiment promotes the expression of SOCS3 in humans or non-human animals by orally ingesting human or non-human animals hyaluronic acid and / or a salt thereof having an average molecular weight of 500,000 or more. can do. This allows inflammatory diseases (eg, rheumatoid arthritis (RA); asthma; allergic diseases such as rhinitis; vascular disease; thrombosis or harmful platelet aggregation; reocclusion after thrombolysis; reperfusion injury; psoriasis, eczema, Skin inflammatory diseases such as contact dermatitis and atopic dermatitis; diabetes (eg, insulin-dependent diabetes, autoimmune diabetes); multiple sclerosis; systemic lupus lupus erythematosus (SLE); ulcerative colitis, Crohn's disease Inflammatory bowel diseases, such as (enteric enteritis) and cystitis (eg, bowel disease that occurs after colorectal resection and ileal anastomosis); childhood lipostool, non-tropical sprue, enteropathy associated with serum reactive negative arthropathy Diseases involving leukocyte infiltration into the gastrointestinal tract, such as lymphocytic or collagenous colitis, and eosinophilic gastroenteritis; white to other epithelial membrane tissues such as skin, urinary tract, airways and joint synovium Diseases related to sphere infiltration; pancreatitis; mastitis (mammary gland); hepatitis; cholecystitis; cholangitis or periductitis (bile duct and liver tissue); tracheoschiaitis; sinusitis; hypersensitivity pneumonitis Inflammatory disease of the lung that causes cystic fibrosis; collagen disease; sarcoidosis; osteoporosis; osteoarthritis; atherosclerosis; neoplastic disease including neoplastic metastasis or cancerous growth; trauma (enhanced trauma healing); retina Exfoliation, allergic conjunctivitis; certain eye diseases such as autoimmunity, uveitis; Sjogren's syndrome; rejection after organ transplantation (chronic and acute); host versus graft or graft versus host disease; intimal thickening; Arteriosclerosis (including graft arteriosclerosis after transplantation); reinfarction or restenosis after surgery such as percutaneous transluminal coronary angioplasty (PTCA) or percutaneous transluminal recanalization; nephritis Tumor angiogenesis; malignant tumor; multiple bones In patients (human or non-human animals) suffering from myeloma; myeloma-induced bone resorption; sepsis; central nervous injury such as stroke, traumatic brain injury and spinal cord injury; and pathology selected from the group consisting of Meniere's disease Symptoms can be improved. Among them, symptoms of autoimmune diseases such as rheumatoid arthritis (RA) and collagen disease can be significantly improved. Examples of non-human animals include mammals other than humans.

  Oral administration of hyaluronic acid and / or salts thereof having an average molecular weight of 500,000 or more to humans or non-human animals, for example, administration site pain, swelling, edema, which may occur when administered locally It is possible to comprehensively improve inflammatory diseases while preventing the occurrence of redness, heat sensation, seriousness and numbness.

1.3. Foodstuff Moreover, the foodstuff which concerns on one Embodiment of this invention contains the SOCS3 expression promoter which concerns on this embodiment. More specifically, the food according to the present embodiment can contain 0.1 to 100% by mass of the SOCS3 expression promoter according to the present embodiment.

  The food (foodstuffs and beverages) according to this embodiment may be intended to alleviate and improve symptoms of inflammatory diseases as a part of treatment or as an adjunct to treatment by the patient taking it over a long period of time. it can.

  Although the mechanism of action by which ingestion of hyaluronic acid and / or its salt promotes the expression of SOCS3 is not necessarily clear, hyaluronic acid is observed on the intestinal epithelial surface of mice ingested by hyaluronic acid or its salt (See FIG. 2 and FIG. 3), the localization of TLR-4, which is a receptor on the intestinal epithelial surface, and the localization of hyaluronic acid are consistent (see FIG. 4). It is speculated that hyaluronic acid and / or a salt thereof is due to promoting the expression of SOCS3.

  In addition to hyaluronic acid and / or a salt thereof, which are SOCS3 expression promoters, the pharmaceutical according to the present embodiment can contain other raw materials as long as the effects of the present invention are not impaired. Examples of such raw materials are water, excipients, antioxidants, preservatives, wetting agents, thickeners, buffers, adsorbents, solvents, emulsifiers, stabilizers, surfactants, lubricants, Water-soluble polymers, sweeteners, flavoring agents, acidulants, alcohols and the like can be mentioned.

  The dosage form of the pharmaceutical product according to this embodiment is not particularly limited, but when the pharmaceutical product is taken orally, for example, a solid preparation such as a tablet, powder, fine granule, granule, capsule, pill, etc., liquid, suspension Orally administrable agents such as liquids such as sachets, syrups and emulsions.

  The food according to this embodiment contains hyaluronic acid and / or a salt thereof, which is a SOCS3 expression agent, as an active ingredient. Although the aspect of the food is not particularly limited, for example, gum, candy, gummy candy, troche-like food, jelly beverage, cooked rice food, bread making, canned retort, frozen food, side dish, dried food, mayonnaise and other seasonings, General foods such as beverages, confectionery, desserts, and supplements, and general health foods that are permitted to express physiological functions are mentioned. Of these, supplements are preferable because of their convenience. .

  The supplements may be in the form of solids such as tablets, powders, fine particles, granules, capsules (hard capsules, soft capsules), fluids, suspensions, jellies, syrups, etc. Is mentioned.

  By orally ingesting the food, it exerts the effect of promoting the expression of SOCS3 and comprehensively alleviates the symptoms of inflammatory diseases. Moreover, the said foodstuff can also be ingested by adding or mixing to the meal of an inflammatory disease.

  As described above, any food and beverage can contain the food according to the present embodiment. However, in order to alleviate the symptoms of the inflammatory disease, the SOCS3 expression promoter according to the present embodiment is continuously used. Therefore, it is desirable to include the SOCS3 expression promoter according to the present embodiment in foods and beverages that are ingested daily.

  Hyaluronic acid and / or its salt is a biological substance, so even if it is ingested in large amounts, it is considered that there is no side effect or very low, but the amount of hyaluronic acid and / or its salt taken as a pharmaceutical is 10 mg per day The standard can be -1000 mg, preferably 100-500 mg. The number of administrations can be selected once or multiple times per day depending on the symptoms. In addition, the amount of the hyaluronic acid and / or salt thereof of the present invention to be ingested as a food can be 1 to 1000 mg, preferably 15 to 300 mg per day. In the case of a beverage containing the hyaluronic acid and / or salt thereof of the present invention, the SOCS3 expression promoter according to this embodiment is 0.001 to 1% by weight in the beverage, preferably 0.01 to 0.5. It can be contained by weight.

2. Proiotrophin expression inhibitor, pharmaceuticals and foods containing the same, and method for suppressing the expression of pleiotrophin The proiotrophin expression inhibitor according to one embodiment of the present invention is the hyaluronic acid and / or its Contains salt as an active ingredient. That is, the hyaluronic acid and / or salt thereof contained in the pleiotrophin expression inhibitor according to this embodiment is hyaluronic acid and / or a salt thereof having an average molecular weight of 500,000 or more, and the above-mentioned SOCS3 expression promoter The same hyaluronic acid and / or a salt thereof exemplified in the column can be used.

  It has been reported that the expression of pleiotrophin is promoted by the onset of autoimmune encephalomyelitis (Liu X, Mashour GA, Webster HF, Kurtz A, “Basic FGF and FGF receptor1 are expressed in microglia during experimenral autoimmune encephalomyelitis: temporally distinct expression of midkine and pleiotrophin ”, Glia., 24, 390-397, 1998.). It has also been reported that pleiotrophin expression is promoted in inflammatory diseases such as rheumatism (Pufe T, Bartscher M, Petersen W, Tillmann B, Mentlein R, “Expression of pleiotrophin, an embryonic growth and differentiation factor, in rheumatoid arthritis ”, Arthritis Rheum., 48, 660-667, 2003.).

  The proiotrophin expression inhibitor according to one embodiment of the present invention contains hyaluronic acid having an average molecular weight of 500,000 or more and / or a salt thereof as an active ingredient, and thereby, for example, exhibits proiotrophin expression by oral ingestion. Can be suppressed.

  The suppression of proiotrophin expression can be evaluated by, for example, the degree of expression of the proiotrophin gene by the DNA array, as shown in the Examples described later.

  Moreover, what was illustrated as a pharmaceutical and a foodstuff containing the proiotrophin expression inhibitor which concerns on one Embodiment of this invention, and its addition amount (compounding quantity) as said pharmaceutical and foodstuff containing the said SOCS3 expression promoter. Can be mentioned.

  Furthermore, the method for suppressing the expression of pleiotrophin according to one embodiment of the present invention comprises orally ingesting hyaluronic acid and / or a salt thereof having an average molecular weight of 500,000 or more to a human or a non-human animal. Including.

3. Examples Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention is not limited to the examples. In the following examples, the average particle diameter of the powder is a value measured by dispersing the powder in ethanol and using SALD-2000A (manufactured by Shimadzu Corporation).

3.1. Example 1
In Example 1, the hyaluronic acid of the present invention was administered by drinking to MRL mice, and the SOCS3 gene expression by the SOCS3 expression promoter containing the hyaluronic acid and / or salt thereof of the present invention was investigated. MRL mice are pathological model mice that spontaneously develop autoimmune diseases such as rheumatism, and various symptoms are known to occur due to autoimmune diseases.

Fourteen MRL lpr / lpr strain (SPF) female mice (Charles River Japan, Inc.) 14 weeks old (body weight approximately 27-37 g) were used in the experiment. Mice were raised in a breeding room set at 22 ± 3 ° C. and a relative humidity of 50 ± 20%, and a solid feed CE-2 (CLEA Japan, Inc.) for rats and mice was fed as a feed. As shown in Table 1 below, hyaluronic acid prepared by the above-described production method (hereinafter also referred to as “hyaluronic acid prototype”) diluted with distilled water was used as a test substance, which was given freely. The details of the test substance are as follows.
Group 1: distilled water (Otsuka distilled water, manufactured by Otsuka Pharmaceutical Factory)
Group 2: Low molecular weight hyaluronic acid (prepared according to the above production method 1 so that the average molecular weight is 2000 (average molecular weight is in the range of 400 to 20,000, average particle size: 67 μm), prototype A)
Group 3: Hyaluronic acid (prepared according to the above production method 2 to have an average molecular weight of 900,000 (average molecular weight in the range of 600,000 to 1.6 million, average particle size: 75 μm), prototype B)
These test substances were prepared aseptically using a clean bench at room temperature. For the preparation, it was adjusted to a predetermined concentration using distilled water for injection and stored refrigerated until use.

  As the dose of the hyaluronic acid prototype, 200 mg / kg / day, which is expected to be effective when administered with drinking water, was set. The amount of water consumed per day by the mouse was 6 mL / animal as a result of preliminary studies, and the body weight of the MRL mouse was about 33 g. Therefore, the concentration of prototype A and prototype B was set to 1.1 mg / mL.

  In addition, it is known that pathological conditions such as lymphadenopathy spontaneously develop in MRL mice. In this example, administration was started from 14 weeks of age, which is the initial stage of the disease state. The dosing period was a 28-day study considered appropriate for evaluating the efficacy of drinking water.

  After a 28-day intake test, the mice were euthanized and the small intestine, large intestine and cervical lymph nodes were removed. Using these samples, (1) confirmation of SOCS3 gene expression by DNA array, (2) confirmation of SOCS3 gene expression by RT-PCR, (3) localization of hyaluronic acid in the intestine, (4) cervix Lymph node weight was investigated.

(1) Confirmation of SOCS3 gene expression by DNA array About 1 cm of a site (sample) including Peyer's plate was collected from the excised large intestine, and subjected to a DNA array using GeneChipMouse Genome 430 2.0 Array (AFFYMETRIX). The expression of SOCS3 gene was examined.

  Preparation of target to be used for DNA array, hybridization, and analysis of DNA array were performed according to GeneChip Eukaryotic Target Preparation & Hybridization Manual (AFFYMETRIX). More specifically, cDNA for RNA in the sample was synthesized, double-stranded cDNA was purified using GeneChip Sample Cleanup Module Kit (AFFYMETRIX), and GeneChip Expression 3'-Amplefication Reagents for IVT Labeling Kit ( Biotin-labeled cRNA was synthesized by biotin-labeling the double-stranded cDNA using AFFYMETRIX (manufactured by AFFYMETRIX), and biotin-labeled cRNA was purified and fragmented using GeneChip Expression Sample Cleanup Module Kit (manufactured by AFFYMETRIX). Next, hybridization control was added to this sample using GeneChip Eukaryotic Hybridization Control Kit (AF FYMETRIX), and hybridization was performed at 45 ° C. for 16 hours at 60 rpm using GeneChip Hybridization Oven 640 (AFFYMETRIX). . Subsequently, washing and streptavidin-phycoerythrin (molecular probe) staining were performed using GeneChip Fluidic Station 450 (AFFYMETRIX). Furthermore, the DNA array obtained by using a laser scanner (GeneChip Scanner 3000 7G (AFFYMETRIX)) is scanned, and the obtained image is used with dedicated software (GeneChip Operating Software ver. 1.4 (AFFYMETRIX))). And analyzed. As a result, SOCS3 gene enhancement was observed in the prototype B intake group.

The gene sequences of the probes fixed to the DNA array are shown in SEQ ID NOs: 1 to 11. The probe information can be confirmed from the following website.
https://www.affymetrix.com/site/login/login.affx
(5´) TCACTTTTATAAAAATCCACCTCCA (3´) (SEQ ID NO: 1)
(5´) GAGGCTGTCTGAAGATGCTTGAAAA (3´) (SEQ ID NO: 2)
(5´) GAAGATGCTTGAAAAACTCAACCAA (3´) (SEQ ID NO: 3)
(5´) GATGCTTGAAAAACTCAACCAAATC (3´) (SEQ ID NO: 4)
(5´) TTGAAAAACTCAACCAAATCCCAGT (3´) (SEQ ID NO: 5)
(5´) TCAACCAAATCCCAGTTCAACTCAG (3´) (SEQ ID NO: 6)
(5´) CAACCAAATCCCAGTTCAACTCAGA (3´) (SEQ ID NO: 7)
(5´) ACCAAATCCCAGTTCAACTCAGACT (3´) (SEQ ID NO: 8)
(5´) CAAATCCCAGTTCAACTCAGACTTT (3´) (SEQ ID NO: 9)
(5´) TCAACTCAGACTTTGCACATATATT (3´) (SEQ ID NO: 10)
(5´) ACTCAGACTTTGCACATATATTTAT (3´) (SEQ ID NO: 11)

  The results of examining the expression of the SOCS3 gene are summarized in Table 2. In Table 2, the expression relative ratio of each group is the fluorescence intensity of each group with respect to the fluorescence intensity of the distilled water intake group, and the fluorescence intensity (average value) by the DNA array is relative to each probe of SEQ ID NOs: 1 to 11. It is an average value of fluorescence intensity.

  As shown in Table 2, the SOCS3 gene expression was approximately doubled in the group administered with prototype B (hyaluronic acid with an average molecular weight of 900,000) as compared to mice that had been drinking distilled water. On the other hand, in the group administered with prototype A (hyaluronic acid having an average molecular weight of 2000), SOCS3 gene expression was suppressed. Therefore, it was suggested that the expression of SOCS3 is promoted by oral ingestion of a hyaluronic acid prototype having an average molecular weight of 500,000 or more.

(2) Confirmation of SOCS3 gene expression by RT-PCR From the excised large intestine, a site (sample) containing Peyer's plate is collected, mRNA is extracted, and RT-PCR using OneStep RT-PCR Kit (QIAGEN) Then, the expression of SOCS3 gene was examined. First, cDNA for mRNA in the sample was synthesized by reverse transcription, and this was amplified by PCR reaction. Specifically, mRNA was first reverse-transcribed at 50 ° C. for 30 minutes, and then PCR was initially activated at 95 ° C. for 15 minutes. Next, the DNA was denatured at 92 ° C. for 30 seconds, annealed at 60 ° C. for 30 seconds, and extended at 72 ° C. for 30 seconds. The above denaturation, annealing, and extension were performed for 30 cycles, and the final extension conditions were 72 ° C. and 10 minutes, and DNA was amplified. This sample was subjected to electrophoresis using 1.5% agarose / TAE buffer and stained with ethidium bromide. In addition, the oligonucleotide which has the gene sequence shown by sequence number 12 and 13 was used as a primer.
(5´) TAGACTTCACGGCTGCCAAC (3´) (SEQ ID NO: 12)
(5´) TCGCTTTTGGAGCTGAAGGT (3´) (SEQ ID NO: 13)

  The results of examining the expression of the SOCS3 gene are summarized in Table 3. The results of electrophoresis are shown in FIG. In Table 3, Relative Optical Intensity is the average value of Relative Optical Intensity for each probe of SEQ ID NOs: 12 and 13.

  As shown in Table 3 and FIG. 1, the SOCS3 gene expression was about 1.3 times higher in the group administered with prototype B (hyaluronic acid with an average molecular weight of 900,000) than in mice that had received distilled water. It was. On the other hand, the group to which prototype A (hyaluronic acid having an average molecular weight of 2000) was administered had SOCS3 gene expression equivalent to that of the distilled water administration group. Therefore, it was suggested that the expression of SOCS3 is promoted by oral ingestion of a hyaluronic acid prototype having an average molecular weight of 500,000 or more.

(3) Localization of hyaluronic acid in the intestine Localization of hyaluronic acid was examined using the excised large and small intestines.

(3-1). Staining of Large Intestine and Small Intestinal Epithelium Hyaluronic Acid A MRL mouse colon specimen was cut to a thickness of 6 μm and air-dried for 30 minutes. This sample was taken out by being immersed in a 10% neutral buffered formalin solution for 30 minutes and washed with PBS for 5 minutes. This washing step was repeated three times, and then pretreatment was performed by immersing in 100 mM acetate buffer (pH 6.0) at 37 ° C. for 15 minutes. The sample after the pretreatment was immersed in a 100 mM acetate buffer (pH 6.0) containing 200 TRU / mL hyaluronidase (derived from actinomycetes, Amano enzyme) at 60 ° C. for 120 minutes to fragment hyaluronic acid. This was washed with PBS for 5 minutes, and this washing step was repeated three times. This was treated with Streptavidin-biotin Blocking Kit (VECTOR) to block endogenous biotin. Specifically, it was immersed in a streptavidin solution at room temperature for 15 minutes, then washed 3 times with PBS for 5 minutes, further immersed in a biotin solution for 15 minutes, and then washed 3 times with PBS for 5 minutes. . After this endogenous biotin blocking treatment, it was immersed in 1% BSA / PBS for 60 minutes. This was immersed in a 1 μg / mL solution (1% BSA / PBS) of HABP-Biotin (Seikagaku Corporation) overnight at 4 ° C., and washed 3 times with PBS for 5 minutes.

  This sample was contacted with Streptavidin HRP at room temperature for 60 minutes, washed 3 times with PBS for 5 minutes, and further contacted with DAB. After this was washed with distilled water, color development was confirmed.

(3-2). Double staining of hyaluronic acid and receptor (TLR-4) of colorectal epithelium (staining method of hyaluronic acid)
Using the sample of the colon epithelium, the HABP-Biotin treatment was performed by the method described in (3) -1. This was immersed in a 1600-fold diluted solution (1% BSA / PBS) of AlexaFluor488 (Moleculer Probe) for 60 minutes at room temperature under light shielding.

(Method for staining TLR-4)
The following procedures were all performed under light shielding. First, the specimen after hyaluronic acid staining was washed three times with PBS for 5 minutes, and then immersed in 10% Donkey serum / PBS for 60 minutes for blocking. This was immersed in 4 μg / mL anti-TLR-4 antibody (10% Donkey serum / PBS) at 4 ° C. overnight, and then washed 3 times with PBS for 5 minutes. This was immersed in a 400-fold dilution (10% Donkey serum / PBS) of AlexaFluor594 (MoleculerProbe), and immersed for 60 minutes at room temperature. This was washed 3 times with PBS for 5 minutes.

(Shooting method)
Using Leica DMI4000B, specimens were photographed at 488 nm (HA) and 594 nm (TLR-4) to confirm the localization of HA and TLR-4.

  As shown in FIG. 2 and FIG. 3, it can be seen that hyaluronic acid is present on the epithelial surface of the small intestine and large intestine of the group administered with prototype B (hyaluronic acid having an average molecular weight of 900,000) (in FIG. 2 and FIG. 3). The arrow indicates hyaluronic acid). Further, as a result of double staining of hyaluronic acid and TLR-4 on the surface of the intestinal epithelium, it was found that the localization of hyaluronic acid was similar to that of TLR-4 (see FIG. 4). From this phenomenon, the ingestion of hyaluronic acid prototypes with an average molecular weight of 500,000 or more promotes the expression of SOCS3 by binding hyaluronic acid to the hyaluronic acid receptor present on the surface of the intestinal epithelium, thereby promoting the expression of pleiotrophin. Presumed that expression is suppressed.

(4) Weight measurement of cervical lymph node Weight measurement and photography of the extracted cervical lymph node were performed. Table 4 shows the results of weight measurement of the cervical lymph node, FIG. 1 shows a photograph of the cervical lymph node, FIG. 2 shows a photograph of the surface of the small intestine epithelium, and FIG. 5 shows a photograph of the surface of the large intestine epithelium.

  As shown in Table 4, the weight of the cervical lymph node of the group to which the prototype B (hyaluronic acid having an average molecular weight of 900,000) was administered, compared with the weight of the cervical lymph node of the group of mice drinking distilled water, It was about 60%, which was significantly small (p = 0.05, Fiche multiple comparison test). On the other hand, the weight of the cervical lymph node in the group administered with prototype A (hyaluronic acid having an average molecular weight of 2000) did not decrease significantly compared to the weight of the cervical lymph node in the group of mice drinking distilled water. As is clear from FIG. 1, it can be seen that the swelling of the cervical lymph nodes of the group administered with the prototype B (hyaluronic acid having an average molecular weight of 900,000) was suppressed. From this phenomenon, it is surmised that the expression of SOCS3 is promoted by oral ingestion of a hyaluronic acid prototype having an average molecular weight of 500,000 or more, thereby reducing the symptoms of inflammatory diseases (autoimmune diseases).

3.2. Example 2
In Example 2, HT29 cells, which are cell lines derived from human large intestine epithelium, were used, and the SOCS3 gene expression by the SOCS3 expression promoter containing hyaluronic acid and / or a salt thereof of the present invention was investigated.

  HT29 cells were cultured under the following conditions.

Prototype A and prototype B used in Example 1 were used and added so that the hyaluronic acid concentration in the medium was 100 ng / mL. After culturing for 24 hours, it was subjected to RT-PCR in the same manner as in Example 1. In addition, the oligonucleotide which has a gene sequence shown by sequence number 14 and 15 was used as a primer, and the amplification cycle was 38 cycles.
(5´) GCCACCTACTGAACCCTCCT (3´) (SEQ ID NO: 14)
(5´) ACGGTCTTCCGACAGAGATG (3´) (SEQ ID NO: 15)

  The results are shown in FIG.

  From the results of FIG. 6, it is understood that the expression of SOCS3 is promoted by culturing HT29 cells in the presence of hyaluronic acid having an average molecular weight of 900,000. On the other hand, in the presence of hyaluronic acid having an average molecular weight of 2000, it can be seen that the expression of SOCS3 is not promoted as compared with the control. From the above, it was suggested that in the presence of hyaluronic acid having an average molecular weight of 500,000 or more and / or a salt thereof, the expression of SOCS3 is promoted in colonic epithelial cells.

3.3. Example 3
Using the prototype B of Example 1 (hyaluronic acid having an average molecular weight of 900,000) as an SOCS3 expression promoter as it was, soft capsules having the following composition were produced.

<Combination ratio>
SOCS3 expression promoter (Prototype B of Example 1) 20%
50% olive oil
Beeswax 10%
Medium chain fatty acid triglyceride 10%
Emulsifier 10%
――――――――――――――――――――――――――――――
100%

3.4. Example 4
Prototype B of Example 1 (hyaluronic acid having an average molecular weight of 900,000) was used as it was as an SOCS3 expression promoter to prepare a powder (granule) having the following composition.

<Combination ratio>
SOCS3 expression promoter (Prototype B of Example 1) 10%
Lactose 60%
Corn starch 25%
Hypromellose 5%
――――――――――――――――――――――――――――――
100%

3.5. Example 5
Prototype B of Example 1 (hyaluronic acid having an average molecular weight of 900,000) was used as it was as an SOCS3 expression promoter to produce tablets having the following composition.

<Combination ratio>
SOCS3 expression promoter (Prototype B of Example 1) 25%
Lactose 24%
20% crystalline cellulose
Corn starch 15%
Dextrin 10%
Emulsifier 5%
Silicon dioxide 1%
――――――――――――――――――――――――――――――
100%

3.6. Example 6
About the same sample as the sample evaluated in Example 1, pleiotrophin gene expression was confirmed by the same method as the confirmation of SOCS3 gene expression by the DNA array of Example 1.

The gene sequences of the probes fixed to the DNA array are shown in SEQ ID NOs: 16 to 26. The probe information can be confirmed from the following website.
https://www.affymetrix.com/site/login/login.affx
(5´) AATGTATACCATAGTACCAGTAGGG (3´) (SEQ ID NO: 16)
(5´) AGGAAGTTGAACTCTGTAGTACATA (3´) (SEQ ID NO: 17)
(5 ′) GATTGAGGTAAGTTTTTTGGTGTTG (3 ′) (SEQ ID NO: 18)
(5´) GTGATATTTCACATTTAAATCTTTT (3´) (SEQ ID NO: 19)
(5´) ATGTTTTCTCTTGTGCATCAATTTA (3´) (SEQ ID NO: 20)
(5´) GTGCATCAATTTAAATGTTACAACC (3´) (SEQ ID NO: 21)
(5´) AACCATGTAAACTACTTCTCTTGTT (3´) (SEQ ID NO: 22)
(5´) AAACTACTTCTCTTGTTAGATAGAT (3´) (SEQ ID NO: 23)
(5´) GATAGATTTTCACCTAGACTTTTTT (3´) (SEQ ID NO: 24)
(5 ′) AGAGGCAGAGCAACGATGTAGTGAA (3 ′) (SEQ ID NO: 25)
(5 ′) AACATGAAATCCTTTCACTTTGGCA (3 ′) (SEQ ID NO: 26)

  In Table 5, the fluorescence intensity (average value) by the DNA array is an average value of fluorescence intensity for each probe of SEQ ID NOs: 5 to 16.

  According to Table 5, it turns out that the expression of pleiotrophin is suppressed by ingestion of the prototype B.

3.7. Example 7
By promoting the expression of SOCS3, it is expected that cytokine signaling is inhibited and the expression of inflammatory cytokines is suppressed. Therefore, the expression of inflammatory cytokines shown in Table 6 was investigated by performing cytokine array using the large intestine of mice ingesting hyaluronic acid according to the present invention (prototype B used in Example 1).

  Using 10 μL of serum collected from the mouse in Example 1 using RayBio Mouse Cytokine Antibody Array 3 (RayBiotech, Inc.), the expression of inflammatory cytokines shown in Table 6 was examined. More specifically, serum was first brought into contact with a glass slide carrier to which antibodies against various cytokines derived from mice were bound. Thereby, antigen-antibody reaction between various cytokines in serum and the antibody occurs. Then, after contacting various anti-cytokine antibodies (secondary antibodies) derived from biotin-labeled mice, streptavidin-fluorescent dye was further added and bound to the secondary antibodies, and the fluorescence intensity was measured. The expression of various cytokines was confirmed. The fluorescence intensity was calculated as an expression ratio with a positive control as a control. The results are shown in Table 6.

  According to Example 1 and Example 2 above, it can be understood that the intake of prototype B promotes the expression of SOCS3 and can reduce inflammatory diseases. Moreover, according to the present Example, it turns out that the expression of IL-10 which is an anti-inflammatory cytokine was accelerated | stimulated by ingestion of the hyaluronic acid (prototype B) which concerns on this invention (refer Table 6). From these results, the intake of prototype B promotes the expression of SOCS3 or suppresses the expression of pleiotrophin, thereby promoting the expression of anti-inflammatory cytokine (IL-10) and reducing inflammatory diseases. It is guessed that it was possible.

  On the other hand, according to Table 6, expression of inflammatory cytokines (MCP-5, MIP-2, P-Selectin, RANTES, SCF, VEGF) was reduced by ingestion of hyaluronic acid according to the present invention (prototype B). . From this result, it can be understood that the expression of inflammatory cytokines was suppressed because inflammation was suppressed by ingestion of hyaluronic acid (prototype B) according to the present invention.

3.8. Example 8
When hyaluronic acid is dissolved in gastric juice at low pH, the molecular weight of hyaluronic acid progresses due to hydrolysis, and the molecular weight of hyaluronic acid may decrease compared to the molecular weight when taken orally. The improvement effect may be reduced.

  Therefore, in this example, the difference in solubility due to the difference in the average particle diameter of hyaluronic acid was evaluated.

3.8.1. Evaluation Method The dissolution rate in artificial gastric juice was evaluated using hyaluronic acid with an average molecular weight of 800,000 having different average particle diameters. Specifically, hyaluronic acid having an average particle size shown in Table 7 was prepared by sieving hyaluronic acid having an average molecular weight of 800,000.

  Using hyaluronic acid having an average molecular weight of 800,000, sieving is performed by sieving 1 (65 mesh (aperture 208 μm)), sieving 2 (150 mesh (aperture 104 μm)), sieving 3 (250 mesh (aperture 61 μm)) ) Sieve was used.

  The particles that passed through the sieve 1 but did not pass through the sieve 2 were used as the particles of Test Example 1, the particles that passed through the sieve 2 but did not pass through the sieve 3 were used as the particles of Test Example 2, and did not pass through the sieve 1 These particles were used as particles of Test Example 3, and particles that passed through sieve 3 were used as particles of Comparative Test Example 1.

  Further, hyaluronic acid having an average molecular weight of 8,000 was similarly sieved to prepare hyaluronic acid having the respective average particle diameters shown in Table 8 (the method for preparing the particles in Reference Examples 1 to 4 is a test example, respectively). 1 to 3 and Comparative Test Example 1).

  0.3 g of each hyaluronic acid was added to 100 mL of artificial gastric juice (NaCl 0.2% aqueous solution of pH 1.2), stirred at the same time, and the time (dissolution time) until complete dissolution was visually measured. Tables 7 and 8 show the dissolution times of hyaluronic acid in Test Examples 1 to 3, Comparative Test Example 1 and Reference Examples 1 to 4, respectively.

3.8.2. Evaluation Results From the above results, the solubility of the hyaluronic acid of Test Examples 1 to 3 (average particle size of 50 to 500 μm) in gastric juice is lower than that of Comparative Test Example 1 (average particle size of less than 50 μm). I understand that. Furthermore, the solubility of hyaluronic acid (average particle size of 80 to 500 μm) in Test Examples 1 and 3 is lower, and the solubility of hyaluronic acid (average particle size of 200 to 500 μm) of Test Example 3 is even lower. From this, the hyaluronic acid of Test Examples 1 to 3 having an average particle size of 50 to 500 μm is less susceptible to hydrolysis in gastric juice than the hyaluronic acid of Comparative Test Example 1 having an average particle size of less than 50 μm, and is low It can be seen that it is difficult to molecularize.

  On the other hand, in Reference Examples 1 to 4 where the average molecular weight of hyaluronic acid is 8,000, it can be seen that the correlation between the average particle size and the dissolution rate is not large. From this, hyaluronic acid having an average molecular weight of 500,000 or more depends on the average particle diameter for solubility in gastric juice, whereas hyaluronic acid having an average molecular weight of less than 500,000 has an average solubility in gastric juice. It can be said that it does not depend on the particle size. In addition, it is shown in the above Example 1 that hyaluronic acid having a molecular weight of less than 500,000 has a lower therapeutic effect on inflammatory diseases than hyaluronic acid having a molecular weight of 500,000 or more.

  As described above, hyaluronic acid having an average particle size of 200 to 500 μm and having an average molecular weight of 500,000 or more is not easily hydrolyzed by gastric juice and is not easily reduced in molecular weight. Therefore, it is presumed that the therapeutic effect of inflammatory diseases is excellent.

Claims (4)

Containing hyaluronic acid having an average molecular weight of 500,000 or more and / or a salt thereof as an active ingredient ,
Treatment of lymphadenopathy associated with rheumatoid arthritis for oral administration , in which the expression of SOCS3 is promoted by ingestion by humans or non-human animals, resulting in suppression of lymphadenopathy that is a symptom of rheumatoid arthritis Agent. The therapeutic agent for lymphadenopathy associated with rheumatoid arthritis for oral administration according to claim 1, wherein the average particle size of the hyaluronic acid and / or salt thereof is 50 to 500 µm. A pharmaceutical comprising the therapeutic agent for lymphadenopathy associated with rheumatoid arthritis for oral administration according to claim 1 or 2 as an active ingredient. The pharmaceutical product according to claim 3 , wherein 10 to 1000 mg is taken per day.