KR102293306B1 - Anti-inflammatory composition for prevention or treatment comprising ion-emitted bioactive glass nanoparticle - Google Patents

Abstract

The present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising ion-releasing mesoporous bioactive glass nanoparticles (BGn) containing calcium (Ca) and silica (Si) as an active ingredient, and the It relates to a method of treating an inflammatory disease using the pharmaceutical composition. The mesoporous bioactive glass nanoparticles provided in the present invention can suppress the expression of COX-2 and iNOS, which are known as inflammation-related factors, by emitting calcium (Ca) and silica (Si) by themselves even without a separate drug. Therefore, it will be widely used for more effective prevention or treatment of inflammatory diseases.

Description

Anti-inflammatory composition for prevention or treatment comprising ion-emitted bioactive glass nanoparticles

The present invention relates to an anti-inflammatory pharmaceutical composition comprising ion-releasing bioactive glass nanoparticles, and more specifically, the present invention relates to an ion-releasing mesoporous bioactive glass containing calcium (Ca) and silica (Si). It relates to a pharmaceutical composition for preventing or treating inflammatory diseases comprising nanoparticles (Bioactive Glass nanoparticles; BGn) as an active ingredient, and a method of treating inflammatory diseases using the pharmaceutical composition.

Inflammation is a complex biological response of the immune system to clear harmful extrinsic and endogenous signals or pathogens and initiate the healing process. Various infectious factors such as bacteria and viruses, ultraviolet light, heavy metals, cholesterol, asbestos and many nanomaterials cause inflammation. Blood monocyte-derived macrophages play an important role in initiating and proliferating inflammatory responses by producing a wide range of inflammatory mediators such as cytokines such as nitric oxide (NO), prostaglandin E2 (PGE2), and interleukin. do. Typically, interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) were shown to increase the inflammatory response. Although the inflammatory response of macrophages and other immune cells is essential for recovery during infection or wound healing processes, permanent macrophage activation has been shown to exacerbate outcomes. In addition, uncontrolled inflammatory responses or chronic inflammatory processes can lead to autoimmune diseases such as inflammatory bowel disease, multiple sclerosis, rheumatoid arthritis, atherosclerosis, chronic hepatitis, pulmonary fibrosis and heart disease.

Synthetic materials have been developed for repair and regeneration of damaged tissue. However, the implanted material is easily recognized as a foreign material and causes a series of inflammatory reactions. In particular, various nano-sized materials such as silica nanoparticles, gold nanoparticles, silver nanoparticles, ceria nanorods, carbon nanotubes and polystyrene nanospheres have been shown to induce inflammatory and immune responses in vitro and in vivo. Therefore, many studies have been made to improve the properties of the material to be more immune-resistant and anti-inflammatory. Interestingly, material parameters including porosity, stiffness, and nanophotography have been reported to alter the inflammatory response. For example, aligned nanofiber scaffolds have been shown to reduce mononuclear cell adhesion and foreign body responses. In addition, surface modification with peptides, nanoparticles, extracellular matrix (eg, fibronectin, vitronectin and laminin) or cytokines has been reported to alleviate inflammation.

Bioactive glass (BG) is known as a biocompatible material widely used to repair bones and surrounding tissues. BG has excellent aggregate conductivity, osteoinductivity and mineralization ability to form a strong bond with bone. In addition, BG can release ions (eg calcium, silica, phosphate and other doped ions) that can promote angiogenesis and wound healing and inhibit the inflammatory response. BG ion extracts treated with endothelial cells have been shown to promote wound healing and prevent apoptosis through modulation of gap junctions. These findings suggest an important role for BG in the inflammatory response and consequent wound healing process.

Among the various types of BG, the nanoparticle form, i.e. BG nanoparticles, has recently attracted attention due to its unique potential as i) nanocarriers of therapeutic molecules, including genes and small molecules, and ii) nanocomponents of bioactive complexes. are getting BGNs currently developed for delivery systems are mainly composed of silica networks with integrated calcium ions and are very mesoporous. Several types of therapeutic molecules, including genes and drugs, have been used to stimulate adult stem cells to differentiate into osteogenic lineages or to inhibit the function of osteoclasts. Although the regenerative potential of BGN has been implicated, the biological action of inflammatory or immune responses remains to be elucidated and is of great importance for understanding the relayed repair processes of inflamed tissues.

Under this background, the present inventors have made intensive research efforts so that bioactive glass nanoparticles (BGn) can be used for inflammation or immune response. As a result, mesoporous bioactivity containing calcium (Ca) and silica (Si) By developing glass nanoparticles and releasing ions without additional drug loading, it inhibits the expression of COX-2 (cyclooxygenase-2) and iNOS genes, reduces the level of inflammatory cytokines, and induces the conversion of macrophages to the phenotype M2. It was confirmed that there was an anti-inflammatory effect, and the present invention was completed.

Republic of Korea Patent Registration No. 10-1972309 Republic of Korea Patent Publication No. 10-2016-0046570

The main object of the present invention is to prevent or treat inflammatory diseases comprising ion-releasing mesoporous bioactive glass nanoparticles (BGn) containing calcium (Ca) and silica (Si) as an active ingredient. to provide a composition.

Another object of the present invention is to provide a method for treating an inflammatory disease using the pharmaceutical composition.

One embodiment of the present invention for achieving the above object is an inflammatory containing ion-releasing mesoporous bioactive glass nanoparticles (BGN) containing calcium (Ca) and silica (Si) as an active ingredient It provides a pharmaceutical composition for preventing or treating a disease.

The present inventors have found that mesoporous bioactive glass nanoparticles (BGN) can be provided as an anti-inflammatory pharmaceutical composition. In particular, the mesoporous bioactive glass nanoparticles (BGN) of the present invention use calcium (Ca) and silicon (Si) as active ingredients to release Ca and Si ions without a separate drug loading, thereby preventing or treating inflammatory diseases. It can be used as a composition. Specifically, through in vivo experiments, it was confirmed that inflammatory cells and the BGN had an anti-inflammatory effect through biochemical interaction with ions released from nanoparticles rather than physical interaction at both the gene and protein level.

As used herein, the term "Bioactive glass (BG)" refers to a glass component capable of inducing a specific biological action in a living tissue, and generally refers to glass composed of inorganic materials. In particular, in the present invention, the bioactive glass can be interpreted as meaning a bioactive glass having mesoporosity in the form of nanoparticles.

As used herein, the term "bioactive glass nanoparticles (BG nanoparticles, BGN)" refers to nanoparticles composed of inorganic components such as silicon, calcium, and phosphorus, wherein the BGN is a compound containing calcium and silicon in a CTAB solution. By adding and reacting the compound containing the compound, an aggregate in which CTAB, calcium and silicon are aggregated is obtained, and the aggregate may be sintered to prepare the aggregate.

Specifically, a calcium oxide precursor is added to a solution of hexadecyltrimethylammonium bromide (CTAB) dissolved in distilled water as a surfactant and mixed, followed by sonicating and stirring while adding a silica precursor to prepare a reaction product, Mesoporous bioactive glass nanoparticles can be obtained by centrifuging, washing, drying, and calcining.

In this case, as the calcium oxide precursor, calcium nitrate tetrahydrate, calcium chloride, calcium acetate, calcium methoxyethoxide, or a mixture thereof may be used, and the like, and the silica Precursors include tetraethyl orthosilicate (TEOS), trimethoxy orthosilicate (TMOS), (3-glycidoxypropyl)methyldiethoxysilane (GPTMS), 3-mercaptopropyl trimethoxysilane (MPS), γ-glycidyloxypropyl trimethoxysilane (GOTMS), aminophenyl trimethoxysilane (APTMOS), or a mixture thereof. can be used The silica precursor solution is preferably prepared by dissolving the silica precursor in an alcohol solution of anhydrous methanol, ethanol, propanol or isopropanol.

In addition, the ultrasonic treatment is preferably performed for 15 to 25 minutes in an output power of 200 W to 240 W, 10 s on / 10 s off cycle.

The calcination may be performed for 8 hours to 12 hours at a temperature range of 500° C. to 600° C. under atmospheric conditions.

The mesoporous bioactive glass nanoparticles prepared by the above method _{include, but are not limited to, silica (SiO 2} ) and calcium oxide (CaO) in a molar ratio of 60:40 to 95:5. For example, the molar ratio of silica to calcium may be 90:10 to 20:80.

The mesoporous bioactive glass nanoparticles provided in the present invention may have a form in which folic acid is bound to the surface thereof.

According to a recent study, when an anti-inflammatory agent was combined with 'folic acid', it was reported that the targeting efficiency and anti-inflammatory activity were significantly increased. Accordingly, when folate is bound to the BGN surface, folate receptors (FRs) are overexpressed on the surface of activated cells and expressed at a low level in normal cells, so folate can be used as a target ligand for inflammatory cells. .

To bind folic acid to the BGn surface, the amination mesoporous bioactive glass nanoparticles were mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) 0.10 to 0.20 M with a cooled folic acid solution. It can be prepared by adding it to the prepared solution.

The average pore size of the mesoporous bioactive glass nanoparticles synthesized by the above method may have a diameter of 150 to 250 nm, but is not limited thereto. For example, it may be 150 to 200 nm.

Mesoporous bioactive glass nanoparticles emit 'calcium ions' or 'silica ions', and the amount of calcium ions released is 500 to 800 ppm based on 50 mg of mesoporous bioactive glass nanoparticles , and the amount of silica ions released is 150 to 300 ppm. can, but is not limited thereto. For example, calcium ions may be 650 to 700 ppm and silica ions may be 200 to 240 ppm. In addition, the release period of the ions may last more than 5 days, but is not limited thereto. For example, it can last up to 7 days.

As used herein, the term “inflammatory disease” refers to a disease in which inflammation is the main lesion. Although not particularly limited thereto, specific examples include erythematosus, atopy, rheumatoid arthritis, Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type 1 diabetes, lupus, chronic fatigue syndrome, fibromyalgia, hypothyroidism and hyperthyroidism, and scleroderma. , Behcet's disease, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Meniere's syndrome, Guilian-Barre syndrome, Sjogren's syndrome, vitiligo, endometriosis, It may be psoriasis, vitiligo, systemic scleroderma, etc., but is not limited thereto.

As used herein, the term “prevention” refers to any action that inhibits or delays the onset of an inflammatory disease by administration of the pharmaceutical composition according to the present invention.

As used herein, the term “treatment” refers to any action in which the symptoms of an inflammatory disease suspect and affected individual are improved or beneficially changed by administration of the pharmaceutical composition.

Persistent inflammation is not beneficial and is involved in the pathology of various diseases, including cancer. Microbial infection or tissue damage activates an inflammatory response, which in turn induces the expression of pro-inflammatory proteins. Inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2) are key enzymes implicated in various inflammatory diseases. iNOS is induced by infection or pro-inflammatory stimuli in a variety of cells. iNOS is most readily observed in macrophages and monocytes in patients with inflammatory diseases. Therefore, iNOS is a very important therapeutic target for the treatment of chronic inflammatory diseases. COX-2 promotes the conversion of arachidonic acid to prostanoids and is an important mediator of inflammatory responses. COX-2 is an inducible enzyme that responds to physical, chemical and biological stimuli and is expressed in most normal mammalian tissues.

By confirming that the mesoporous bioactive glass nanoparticles according to the present invention inhibit the expression of COX-2 and iNOS, which are known as inflammation-related factors, it was confirmed that they can be used for the prevention or treatment of inflammatory diseases.

Treatment with the compositions of the present invention can reduce the expression of representative proinflammatory cytokines. Specifically, it may decrease the secretion or expression of cytokines of CCL4/MIP-1β, GM-CSF, IFN-γ, IL-1α, IL-4, IL-6 and/or TNF-α, but is not limited thereto. does not Furthermore, it is possible to relieve inflammation by converting M1 macrophages to M2 type during the inflammatory response.

The "expression" includes both gene expression and protein expression.

The “M1 macrophage” is an inflammatory macrophage that removes cell debris caused by damage and swallows and digests damaged necrotic tissue. It also secretes growth factors and cytokines necessary for cell proliferation, migration and differentiation. However, if M1 macrophages persist, they can lead to cell and tissue damage by the release of inflammatory cytokines and cytotoxic molecules such as ROS, leading to secondary damage and dysfunction. On the other hand, anti-inflammatory M2 macrophages in damaged tissues can attenuate inflammation and promote tissue repair processes.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent, which may include a non-naturally occurring carrier.

More specifically, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate , calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, polycaprolactone, polylactic acid (Poly Lactic Acid), poly-L-lactic acid, mineral oil, and the like.

The pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., oral dosage forms, external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. The carrier may include various amorphous carriers, microspheres, nanofibers, and the like.

In the case of formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract and its fractions, for example, starch, calcium carbonate, It may be prepared by mixing sucrose or lactose, gelatin, or the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Liquid formulations for oral administration include suspensions, solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have.

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

The content of the mesoporous bioactive glass nanoparticles included in the pharmaceutical composition of the present invention is not particularly limited thereto, but based on the total weight of the final composition, 0.0001 to 80% by weight, 0.0001 to 50% by weight, more specifically 0.01 to 20 It may be included in a content of wt%.

Another embodiment of the present invention provides a method for preventing or treating an inflammatory disease, comprising administering the composition to an individual other than a human having an inflammatory disease or a risk of developing an inflammatory disease.

In this case, the descriptions of the “inflammatory disease”, “mesoporous bioactive glass nanoparticles”, “prevention” and “treatment” are the same as described above.

Since the pharmaceutical composition of the present invention exhibits a preventive or therapeutic effect on an inflammatory disease, the method of the present invention comprising administering it to an individual can be usefully utilized for the prevention or treatment of an inflammatory disease.

As used herein, "administration" means introducing the composition to a subject by an appropriate method.

As used herein, the term "individual" refers to all animals, such as rats, mice, and livestock, including humans, that have or can develop an inflammatory disease, and specifically, may be mammals including humans, but is not limited thereto. .

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, and the term, "pharmaceutically effective amount" is sufficient to treat or prevent a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention. means the amount, and the effective dose level is the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the administration time of the composition of the present invention used, the route of administration and the rate of excretion Treatment period , factors including drugs used in combination with or concurrently with the composition of the present invention and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered alone or in combination with a known therapeutic agent for pterygium. Taking all of the above factors into consideration, it is important to administer an amount capable of obtaining the maximum effect with a minimum amount without side effects.

The pharmaceutical composition of the present invention may be administered using various methods such as oral, intravenous, subcutaneous, intradermal, intranasal, intraperitoneal, intramuscular, and transdermal, and the dosage may depend on the age, sex, and weight of the patient. may vary and can be readily determined by one of ordinary skill in the art.

In addition, the dosage of the pharmaceutical composition can be determined by those skilled in the art in consideration of the purpose of use, the degree of addiction of the disease, the age, weight, sex, history, or the type of substance used as an active ingredient. For example, the pharmaceutical composition of the present invention may be administered at an amount of 1 mg/kg to 200 mg/kg, specifically 1 mg/kg to 100 mg/kg, and more specifically 20 to 40 mg/kg per adult. , The frequency of administration of the composition of the present invention is not particularly limited thereto, but may be administered once a day or administered several times by dividing the dose. The above dosage does not limit the scope of the present invention in any way.

In addition, the dosage of the pharmaceutical composition may be adjusted so that the mesoporous bioactive glass nanoparticles are administered at 80 to 120 mg/kg, more specifically, 100 mg/kg, but is not limited thereto.

The mesoporous bioactive glass nanoparticles provided in the present invention can suppress the expression of COX-2 and iNOS, which are known as inflammation-related factors, by emitting calcium (Ca) and silica (Si) by themselves even without a separate drug. Therefore, it will be widely used for more effective prevention or treatment of inflammatory diseases.

Figure 1 (A) is a transmission electron microscope (TEM) photograph showing the shape of the BGN (F) provided in the present invention. (B) is a graph showing the result of recording the amount of ions (silica silicate and calcium) released by BGN(F) up to 7 days by ICP-AES. (C) is a schematic diagram showing the interaction of BGN (F) with targeted inflammatory active cells. (D) is a graph showing the cell density by fractionation of FITC-positive cells recorded in 4 hours of culture of BGN or BGN (F) bound to FITC analyzed by FITC. (E) is a fluorescence micrograph of the cells treated with BGN (F).
Figure 2 (A) BGN (F) treatment of the change in cell viability and LPS-treated RAW 264.7 cells treated with various concentrations of BGN (F), and the viability was analyzed with CCK-8 (n = 3). As a graph showing, BGN(F) shows the result of performing a comparative experiment with mesoporous silica nanoparticles (MSN) and an ion extract group of BGN(F). (B) is a graph showing the amount of LDH release (%) monitored by the Pierce LDH cytotoxicity assay kit. (C) is a graph showing the ROS generation level analyzed by the Total ROS Detection Kit, RAW 264.7 cells were pretreated with LPS (1 μg/mL) and BGN ( F) Results of incubation with 80 or 160 μg.
3 is a graph showing the results of comparing the expression levels of inflammation-related genes quantitatively analyzed by qRT-PCR. Treatment of BGN(F) 80 or 160 μg in LPS-treated RAW 264.7 cells for 24 hours is inflammation-related. The expression of genes (TNF-α, IL-6, COX-2 and iNOS) was significantly reduced. The effects of ionic extracts of MSN and BGN (F) on cells were also investigated for comparative purposes.
4 is a fluorescence micrograph and graph showing the experimental results regarding the expression of inflammation-related proteins (TNF-α, IL-6, COX-2 and iNOS). (A) is a fluorescence micrograph of cells visualized by immunocytochemical staining by CLSM, in which LPS-treated RAW 264.7 cells were treated with 80 or 160 μg of BGN(F) for 24 h to induce inflammation-related proteins (shown in green). It was confirmed that the level of was significantly reduced, at this time, counterstaining was performed with DAPI (shown in blue). (B) is a histogram showing the FACS analysis result, (C) is a graph showing the analysis result of the histogram, cells without LPS and BGN (F) treatment used as negative control (N Ctrl) and LPS (1 μg /mL) was used as a positive control (LPS). (D) is a graph showing the level of release of inflammatory cytokines from cells monitored by ELISA. LPS-treated cells were cultured with 80 or 160 μg of BGN(F) for 24 hours, and then released TNF-α and IL- 6 was analyzed with an ELISA kit (n = 3). (E) is a graph showing the results of the COX-2 inhibition assay, in which LPS-induced cells were incubated with 80 or 160 μg of BGN (F) for 24 hours and then analyzed with the COX-2 inhibitor screening kit, COX -2 Compared with the control group (N Ctrl), the COX-2 inhibitory activity was 14.38% and 37.45%, respectively (BGN(F) 80 and 160 μg), and cells treated with the COX-2 inhibitor Celexoxib were used as the control group. was used
5 is a graph showing the results of performing a macrophage polarization experiment. (A) is a graph showing the amount of nitric oxide production according to the concentration of BGN(F) in LPS-treated RAW 264.7 cells, (B) is the amount of urea production according to the concentration of BGN(F) in the LPS-treated RAW 264.7 cells is a graph representing
6 shows the results of analyzing the in vivo activity of BGN(F) in a muscle damage (myoinjury) mouse model. (A) is a schematic diagram showing the manufacturing process of a mouse model of a muscle injury that damaged the anterior tibial (TA) muscle of the mouse. (B) is a photomicrograph showing the results of staining the muscle tissue with HE and MT at 3 and 7 days after administration of BGN(F). Negative control group (N Ctrl) without any action, Notexin Alternatively, a positive control group treated with physiological saline (Saline) was used. In the positive control group, muscle fiber necrosis and degeneration were induced, and it was confirmed that the number of inflammatory cells increased. (C) is a fluorescence micrograph showing the immunofluorescence staining results showing the levels of IL-6 and TNF-α in the tissue samples at 3 days and 7 days elapsed, (D) is the IL from the immunofluorescence staining results It is a graph (D) showing the result of analyzing the expression level of -6 or TNF-α. (E) is a fluorescence micrograph showing the results of immunofluorescence staining of inflammatory M1 macrophages showing a CD80 positive reaction and anti-inflammatory M2 macrophages showing a CD163 positive reaction in a time point tissue sample after 3 days, (F) is the It is a graph showing the result of analyzing the expression level of CD80 or CD163 from the results of immunofluorescence staining.
7 is a histogram and graph showing the results of FACS analysis targeting important intracellular inflammatory signaling molecules involved in inflammation (MAPK, ERK (1/2), SAPK / JNK, IkBA and NFkB). Cells treated with LPS was incubated with BGN (F) for 24 hours. (A) is a histogram showing the expression level of each signal molecule by BGN(F) treatment, and (B) is a graph showing the phosphorylation level of each signal molecule.
8 is a schematic diagram showing the therapeutic action mechanism of BGN(F) on a series of inflammation-related signaling processes in LPS-treated macrophages.

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

Example 1. Preparation of bioactive glass nanoparticles (BGN)

Dissolve 1 g of hexadecyl-trimethylammonium bromide (CTAB) in a solution consisting _{of 140 mL of distilled water, 2 mL of NH 4} OH, 40 mL of ethoxyethanol, 20 mL of ethanol and Ca(NO ₃ ) ₂ .4H _{2 O.} It was dissolved, and after vigorous stirring at room temperature for 30 minutes, further tetraethyl orthosilicate (TEOS) was rapidly added dropwise, followed by reaction with stirring at room temperature for 4 hours. After the reaction was completed, centrifugation was performed at 10000 rpm to obtain a white precipitated powder, and the obtained powder was thoroughly washed with pure water and ethanol. The washed powder was dried in air at 60° C. for 24 hours, and then calcined at 550° C. for 10 hours to prepare bioactive glass nanoparticles (BGN) from which residual CTAB was removed.

Example 2. Preparation of folic acid-conjugated bioactive glass nanoparticles (BGN(F))

(3-aminopropyl)triethoxysilane (APTES) was added to the BGN prepared in Example 1 at 80° C. for amination. In addition, 0.15 M of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) was dissolved in 500 μg/mL of a cooled folic acid solution, and then the amination BGN was added at 9 mg/mL. mL, and reacted with stirring at room temperature for 24 hours. After completion of the reaction, the reaction product was centrifuged to obtain a precipitate, and the obtained precipitate was washed with distilled water and then freeze-dried for 48 hours to obtain bioactive glass nanoparticles (BGN(F)) having folic acid bound to the surface. was prepared.

Example 3. Characterization of BGN(F)

First, the nanostructure of BGN(F) synthesized in Example 2 was observed by a transmission electron microscope (TEM; JEM-3010, JEOL, Japan) (FIG. 1A). As shown in FIG. 1A , it was confirmed that nanospheres of uniform size (about 200 nm in diameter) were formed, and a high level of mesoporous structure was exhibited.

Next, the chemical bond structure of BGN(F) was analyzed with reduced total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR, Varian 640-IR) using the GladiATR diamond crystal accessory (PIKE Technologies) with a resolution of ^{4 cm -1 .} analyzed. As a result, it was confirmed that BGN showed ^{Si-O-Si symmetric stretching on silica 1095 cm -1} , Si-O-Si asymmetric stretching on 801 cm ^-1 , and 465 cm ^-1 , and the amination BGN was 695 cm -1 . It ^{was confirmed that NH vibration peaks at cm -1} and 1570 cm ^-1 appeared, and BGN(F) confirmed that the NH vibration peak intensity decreased and amides 1654 and 1562 cm ^-1 appeared, and folic acid was bound to the BGN. was found to be in the form of

Next, the emission of silica and calcium ions from BGN(F) was examined using inductively coupled plasma-atomic emission spectroscopy (ICP-AES, OPTIMA 4300 DV, Perkin-Elmer, USA) for 7 days. That is, 50 mg of nanoparticles were dispersed in 10 mL of Tris buffer solution (pH 7.4) and placed in an incubator shaker operated at 37° C. and 120 rpm. Samples were taken at predetermined time points, centrifuged at 15,000 rpm for 10 minutes, and the supernatant was collected for ion analysis (FIG. 1B). As shown in FIG. 1B , the emission profiles of the two ions are similar. It was confirmed that the release rate progressed relatively rapidly up to the first 3 days and slightly decreased on the 7th day. It was confirmed that the calcium ion release amount was ~699ppm for 7 days, and the silica ion release amount was ~237ppm.

Example 4. Evaluation of the specific receptor binding ability of folic acid and inflammatory cells

The reason for preparing BGN(F) in Example 2 is to make BGN(F) more specifically act on inflammatory cells (FIG. 1C), so to analyze the binding ability of BGN(F) to inflammatory cells. did. Roughly, BGN(F) was labeled with FITC (Sigma) for 1 h to evaluate the uptake efficiency of nanoparticles into LPS-induced inflammation. For this, the cells were seeded in a 6-well plate at 1 x 10 ⁵ / well, and 80 μg of FITC-tagged BGN (F) was added to each well with 1 mL Opti-MEM (Invitrogen, USA). Then incubated for up to 4 hours. After the BGN(F)-treated cells were harvested and fixed with 4% paraformaldehyde (PFA) solution for 1 hour, the intracellular uptake efficiency of nanoparticles was performed using a FACS Calibur flow cytometer (FACS; BD Biosciences, USA). was quantified. Data collected for 10,000 cells in each sample was analyzed using CellQuest Pro software (BD Biosciences) (FIG. 1D). As shown in FIG. 1D , the BGN(F) group showed 2.97, 3.17, 5.25, 12.02, 20.14 and 52.29% at 10, 20, 40, 60 80 and 160 μg/mL, respectively. The BGN group showed 0.85, 0.82, 0.77, 2.11, 2.39 and 6.22% at the corresponding concentrations.

In addition, the FITC signal in the BGN or BGN(F)-treated cells was photographed with a fluorescence microscope (FIG. 1E). As shown in FIG. 1E, it was confirmed that a stronger FITC(+) signal was observed in the cytoplasm when treated with BGN(F) than BGN. When unstimulated RAW cells were treated with the BGN or BGN(F), this difference did not appear, but the difference was clearly shown in the proinflammatory cells, thus confirming that BGN(F) specifically binds to the inflammatory cells. did.

Example 5. Cell viability evaluation

The effect of BGN(F) on the viability of cells was investigated. RAW 264.7 cells induced by LPS were plated in each well of a 96-well plate at a density of 10,000 cells per well and stabilized for 24 hours. The culture medium was then replaced with 1 mL of BGN(F) solution (0, 10, 20, 40, 80 or 100 μg/mL) in DMEM medium. After incubation for 24 hours, cell viability was measured with a CCK-8 cell counting kit (Dojindo, Japan). Cells were washed with PBS and then treated with 10 μL of CCK-8 solution and 100 μM of DMEM medium, and incubated at 37° C. for 4 hours. The CCK-8 solution is a reagent developed to measure cell viability and activity. When treated with cells, the CCK-8 solution is absorbed into cells and binds to mitochondrial metabolites to show a color reaction. activity and survival rate can be measured. After incubation, 100 μL of each cell culture supernatant was collected and absorbance was measured at 450 nm using an iMark microplate reader (BioRad, USA). Absorbance values were normalized to the values of the negative control group without nanoparticles, and the data were averaged from 3 samples (n = 3). Live and dead cell images were taken using a live and dead stain kit (Molecular Probe, USA). For comparative purposes, another type of nanoparticles, mesoporous silica nanoparticles (MSNs), were also used. In addition, ionic extracts of BGN(F) were collected and used for cell analysis. For ion extraction, BGN(F) was incubated with a culture medium in an incubator for 24 hours, and the culture medium was treated with cells by centrifugation (FIG. 2A). As shown in FIG. 2A , the viability of RAW 264.7 cells induced by inflammation was reduced to 64.3% w.r.t by LPS. Accordingly, it was confirmed that LPS was detrimental to cell viability.

Next, as a result of evaluating the effect on RAW 264.7 cells induced by inflammation by LPS treated with BGN(F) at various concentrations for 24 hours, it was found that the decrease in viability was significantly lower than that of LPS-treated cells. That is, it was confirmed that BGN(F) could significantly improve inflammatory cell viability under LPS treatment conditions (82.3% and 79.9% at 80 and 160 μg/mL BGN(F) treatment, respectively).

In addition, the effect of BGN(F) treatment on cells was compared with that of MSN and BGN(F) ion extract treatment. When the ionic extract of BGN(F) was treated to LPS-treated cells, the viability showed that the observed BGN(F) was significantly similar to the level of inflammatory cell viability under LPS-treated conditions (80 and 160 μg/mL). 78.5% and 78.4% with ion treatment). However, when MSN was treated, the viability of LPS-treated cells was not restored. All MSN concentrations showed 50-60% cell viability. This was confirmed to be a survival rate similar to that of about 64.3% in the LPS-treated control group.

Example 6. Comparison of LDH Release and ROS Production

First, LDH released from the cells was measured using the Pierce LDH Cytotoxicity Assay Kit (Thermo Fischer, USA) according to the manufacturer's protocol. That is, cells in a 96-well plate were treated with nanoparticles for 24 hours. After incubation, 50 μL of the supernatant from each well was transferred to a new 96-well plate. After that, LDH assay solution (50 μL) was added to each well, and the samples were incubated in the dark for 30 min. The emitted total LDH was measured at 490 nm (FIG. 2B). Data were expressed as a percentage of the control. As shown in FIG. 2B, it was confirmed that the LDH release level of cells treated with BGN(F) was reduced to 72.1% and 63% at 80 and 160 μg/mL, respectively, compared to the control without BGN(F) treatment. .

In addition, ROS production in cells was analyzed with Total ROS Detection Kit (ENZO, USA). Cells treated with nanoparticles for 24 hours were analyzed for ROS production levels using a multi-detection microplate reader, SperctaMaxM2e (Molecular Devices Corp., USA) according to the manufacturer's protocol (FIG. 2C). As shown in FIG. 2C , BGN(F)-treated cells showed a significantly reduced level of ROS production (80.45% and 66.85% at 80 and 160 μg/mL, respectively).

Example 7. Evaluation of effects on expression of inflammation-related genes and/or proteins

The anti-inflammatory effect of treatment with BGN(F) on LPS-treated cells was investigated. Total RNA was extracted from nanoparticle-treated cells using the RNeasy min kit (Qiagen, USA), and interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α) , The expression of representative inflammation-related genes including cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) was quantitatively analyzed by RT-PCR for 24 hours treated with BGN(F). . In this case, the glycerol aldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an internal control group. Real-time PCR was performed using SYBR qPCR SuperMix reagent (Invitrogen) and Bio-Rad iCycler. The relative transcription amount was calculated using the ΔΔCt method using the GAPDH gene as an endogenous reference gene amplified from each sample. The primer sequences for RT-PCR of each gene are as follows:

IL-6 F: 5'-GAAATGATGGATGCTACCAAACTG-3' (SEQ ID NO: 1)

IL-6 R: 5'-GACTCTGGCTTTGTCTTTCTTGTT-3' (SEQ ID NO: 2)

TNF-α F: 5'-TACTGAACTTCGGGGTGATCG-3' (SEQ ID NO: 3)

TNF-α R: 5'-GGGTCTGGGCCATAGAACTGA-3' (SEQ ID NO: 4)

COX-2 F: 5'-GAAGTTGATAACCGAGTCGTTC-3' (SEQ ID NO: 5)

COX-2 R: 5'-CCATAGAATAACCCTGGTCG-3' (SEQ ID NO: 6)

GAPDH F: 5'-AGGAGCGAGACCCCACTAACA-3' (SEQ ID NO: 7)

GAPDH R: 5'-AGGGGGGCTAAGCAGTTGGT-3' (SEQ ID NO: 8)

iNOS F: 5'-CATGCTACTGGAGGTGGGTG-3' (SEQ ID NO: 9)

iNOS R: 5'-CATTGATCTCCGTGACAGCC-3' (SEQ ID NO: 10)

Ion extract or MSN-treated cells were analyzed and compared with BGN(F)-treated cells ( FIG. 3 ). As shown in FIG. 3 , it was confirmed that all genes were significantly reduced in cells when treated with 80 and 160 μg/mL of BGN(F). In comparison, cells treated with ion extracts labeled 'ion 80' and 'ion 160' showed reduced expression of all inflammatory genes, but the effect was lower than that of BGN(F) treatment. On the other hand, cells treated with MSN at 80 and 160 μg / mL, respectively, showed that MSN had no effect on suppressing inflammatory genes.

In addition, it was applied to a screening assay for examining 15 types of inflammation-related cytokines secreted from inflammatory cells based on inflammation-related genes expressed in BGN (F)-treated cells (Table 1).

Cytokine concentration (μg/mL) Control LPS BGN(F) 80 BGN(F) 160 CCL3/MIP-1α 3.3±0.77 2.79±0.29 3.47±0.22 5.55±2.41 CCL4/MIP-1β 198,765.49±12625.51. 419,447.31±187042.70 232,683.90±26568.29 320,268.05±5507.06 CXCL10/IP-10 692.27±19.60 1,556.67±13.99 1,427.36±53.55 1,275.64±46.99 GM-CSF 106.52±3.20 233.99±6.70 146.49±5.47 363.39±12.60 IFN-γ 51.36±1.54 100.55±1.12 88.26±2.39 91.61±0.01 IL-1α 116.44±0.01 467.51±13.61 327.76±13.45 312.88±10.59 IL-1β 185.03±23.07 233.44±0.01 217.39±22.69 225.45±11.30 IL-10 14.04±0.96 32.15±0.65 27.55±1.95 27.09±1.30 IL-12 p70 49.03±2.22 72.11±8.55 66.03±8.60 72.11±8.55 IL-13 86.13±12.99 104.37±17.96 102.60±5.14 98.04±9.02 IL-2 5.43±0.33 5.19±0.01 7.52±0.66 6.59±1.97 IL-4 77.69±12.19 92.74±3.02 86.30±12.14 86.31±0.01 IL-6 964.68±20.41 1,706.23±37.58 1,199.99±42.77 1,521.97±56.42 IL-6Rα 162.05±52.69 796.08±0.01 732.50±38.37 742.19±128.07 TNF-α 946.92±25.79 1,466.92±7.94 1,045.69±33.89 1,146.72±19.32

As shown in Table 1, the levels of pro-inflammatory cytokines including IL-1α, IL-4, IL-6 and TNF-α were significant in the cells induced by inflammation by BGN(F)-treated LPS. appeared to be significantly reduced

Meanwhile, the cell expression of TNF-α, IL-6, COX-2 and iNOS among the inflammation-related molecules was analyzed by immunocytochemical staining ( FIG. 4 ). At this time, as a negative control (N Ctrl), cells not treated with LPS and BGN(F) were used, and as a positive control, cells treated only with LPS (1 μg/mL) and not treated with BGN(F) were used. did. As shown in FIG. 4 , the inflammatory signal was strongly expressed in LPS-treated cells as visualized under a confocal microscope, but was significantly attenuated in BGN(F)-treated cells ( FIG. 4A ). In addition, it was confirmed that the population of cells expressing TNF-α, IL-6, COX-2 and iNOS protein was also substantially reduced by BGN(F) treatment as shown in the FACS histogram (FIG. 4B). In particular, the cell population positive for the inflammatory signal was significantly reduced at 80 μg/mL of BGN(F). The decreasing pattern of protein expression appeared to be consistent in cells treated with 80 μg/mL compared to cells treated with 160 μg/mL (Fig. 4C).

The time-dependent release of two representative pro-inflammatory cytokines (TNF-α and IL-6) from inflammatory cells was analyzed by ELISA (FIG. 4D). The levels of cytokines released from cells treated with LPS were shown to decrease by 3 days of incubation time. Specifically, it was confirmed that the release of both cytokines was significantly reduced by 30-40% of the control group at both concentrations of 80 μg/mL and 160 μg/mL treated with BGN(F) compared to the LPS-treated control group.

The effect of BGN(F) treatment on COX-2 as a major molecule of proinflammatory action was investigated using a COX-2 screening assay kit. COX-2 production in the positive control group was 45% of that of the untreated control group, and treatment with BGN(F) (80 and 160 μg/mL) was 85.61% (80 μg/mL) and 62.54% compared to the untreated control group. % (160 μg/mL) significantly decreased ( FIG. 4E ).

Cells were treated with BGN for 24 h and polarization of macrophages was measured using Nitric Oxide (NO) Assay Kit (abcam, USA) and urea Assay Kit (Sigma, USA) according to the manufacturer's protocol. analyzed ( FIG. 5 ). Roughly, to assess macrophage polarization, we measured the production of nitric oxide (NO) and urea, which are representative of M1 and M2 macrophages, respectively. As shown in FIG. 5 , it was confirmed that LPS-treated cells produced significantly more nitric oxide and less urea compared to untreated macrophages. In addition, it can be seen that BGN(F) treatment changed the cell phenotype from M1 to M2 by confirming that BGN(F) treatment reduced NO concentration and significantly increased urea concentration compared to LPS-treated cells.

Example 8. Effect of BGN treatment on inflamed muscle tissue (in vivo)

8.1 Experimental method

A rat anterior tibial (TA) muscle injury model was used to investigate the in vivo anti-inflammatory effect of BGN(F) (all experimental procedures were approved by the Animal Care and Use Committee of Dankook University). Seven-week-old 22-26 g healthy male BALB/C mice were used for the experiments. Ketamine (ketamine) 80 mg / kg and xylazine (xylazine) 10 mg / kg was injected into the muscle tissue was carried out under general anesthesia. For muscle injury, 10 μg/ml of Notexin (purchased from Sigma), a neurotoxic and myotoxic phospholipase A2 derived from snake venom, was injected into the left TA muscle (n = 5). The TA muscle of the contralateral leg was used as a negative control. After 24 h, BGN or BGN(F) was injected into TA muscle (300 μg/leg) with damaged myoinjury, and rats were allowed ad libitum normal diet and water while maintaining the barrier system under a controlled environment. did. Animals were sacrificed 3 and 7 days after sample injection and TA muscle tissue was obtained from the surrounding tissue. The TA muscle tissue samples were analyzed after being fixed in 10% neutralizing buffered formalin for 24 hours (FIG. 6A).

Fixed tissue samples were dehydrated with a graded series of ethanol and embedded in paraffin using standard procedures. Paraffin sections cut to a thickness of about 5 μm were deparaffinized, treated with hematoxylin & eosin (HE) and Masson's trichrome (MT), and then observed and imaged using a microscope. HE and MT staining images are visualized for histological examination of the inflamed muscles (Fig. 6B). For immunohistochemical staining of biochemical samples, deparaffinized sections were washed three times with PBS and incubated with IL-6, TNF-α, CD80 (abcam) and CD163 (Bioss) antibodies at 4° C. for at least 24 hours. After rinsing with phosphate-buffered saline (PBS), the sections were incubated with FITC-conjugated secondary antibody (Santa Cruz Biotechnology) in a humidified chamber at room temperature for 1 hour. Nuclei were stained with DAPI. Fluorescence images were observed using a laser scanning microscope (Zeiss LSM 510). For quantification, the fluorescence intensity measured from randomly acquired images of each experimental group was analyzed using Zeiss LSM software. Data were expressed as mean ± 1 standard deviation (SD), statistical analysis was performed by one-way analysis of variance (ANOVA) and P values less than 0.05 were considered statistically significant.

The legs without any treatment and the legs treated only with Notexin were used as negative (N Ctrl) and positive controls (Notexin), respectively, and were treated with saline only, BGN treated with saline, and BGN treated with BGN (F). Experiments were performed with the BGN(F) group.

8.2 Effect of muscle fiber rearrangement and muscle tissue regeneration evaluation

As prepared in Example 8.1, the leg without any treatment and the leg treated only with Notexin were used as negative (N Ctrl) and positive controls (Notexin), respectively, and the group treated with only saline and the group treated with BGN with saline and BGN(F)-treated BGN(F) group (B of FIG. 6).

As shown in FIG. 6B , the negative control group showed a typical muscle tissue structure composed of elongated, multinucleated muscle fibers bound by collagenous support tissue without conspicuous inflammatory markers. In the positive control (Notexin) and saline on day 3, the tissue is mainly filled with degenerative muscle fibers, necrotic tissue and infiltrated inflammatory cells. Tissue fibrosis was also observed by MT staining. However, in the BGN and BGN (F) groups at day 3, myofibers were well preserved and inflammatory cells were substantially reduced around the myotubes derived tissue. On day 7, in the Notexin and Saline groups, the damaged muscle tissue was gradually replaced by small regenerative muscle myofibrils, but degenerative tissue was still observed among the muscle fibers. On the other hand, the BGN and BGN(F) groups showed rearranged and regenerated muscle fibers. Also, myofiber rearrangement and muscle repair were more significant in BGN(F) than in BGN.

8.3 Assessment of Inflammation-Related Protein Expression

Immunofluorescence staining was performed to analyze the intracellular expression levels of IL-6 and TNF-α proteins in in vivo tissues ( FIGS. 6C and 6D ). As shown in FIG. 6C , both protein signals were very strong in the Notexin and Saline groups, but significantly decreased in the BGN and BGN(F) groups. As shown in FIG. 6D, when BGN(F) and BGN were compared, both proteins showed significantly lower levels than BGN in the BGN(F) group.

8.4 Evaluation of macrophage polarization in vivo (in vivo)

In vivo macrophage polarization by treatment with BGN or BGN(F) nanoparticles was analyzed. To this end, on day 3, tissue samples were immunohistochemically stained for CD80 and CD163, which are known surface markers of M1 and M2, respectively ( FIGS. 6E and 6F ). As shown in FIG. 6E , the Notexin and Saline groups showed a strong CD80 positive reaction, but the BGN and BGN(F) groups showed strong CD163 expression. As shown in FIG. 6F , as a result of quantitative analysis of CD80 and CD163, both nanoparticles significantly converted the macrophage phenotype from M1 to M2 in the muscle inflamed tissue, and macrophage polarization also had a higher conversion rate in BGN(F) than in BGN. appear.

Example 9. Anti-inflammatory effect-related intracellular signal transduction pathway by BGN

Expression and phosphorylation levels of various inflammatory signaling molecules including MAPK, ERK (1/2), SAPK/JNK, IkBA and NF-kB were analyzed by FACS ( FIGS. 7A and 7B ).

As shown in A and B of FIG. 7 , when BGN(F) was treated and cultured for 24 hours, phosphorylation of inflammatory signaling molecules was particularly decreased as the concentration of BGN(F) increased. In particular, when treated with 80 μg and 160 μg BGN(F), respectively, compared with the LPS control, the expression of phosphorylation signal molecules was reduced by 81% and 33% for p38 MAPK, and 48% and 26% for ERK(1/2). %, 77% and 34% for SAPK/JNK, 42% and 25% for SAPK/JNK, and 44% and 88% for NF-kB.

As is commonly known, the signaling pathway of LPS-treated cells is initiated by LPS binding to the receptor complex and the NF-κ pathway is activated. In normal cells, NF-κ is constitutively restricted to the cytoplasmic NF-κ inhibitor, Iκ. Upon stimulation with LPS, Iκ protein is phosphorylated, ubiquitinated, and rapidly degraded by the ubiquitin-proteasome pathway, and free NF-κ migrates to the nucleus to activate the transcription of NF-κ-dependent inflammatory enzymes and cytokines. In addition, a family of MAPKs, including Erk1/2, p38 MAPK and JNK, has been shown to play important roles in the mediation of inflammatory signals induced by cytokines, growth factors and environmental stress. In particular, the increased activity of p38 MAPK is strongly involved in the synthesis of inflammatory mediators at the transcriptional and translational level. It is also known that activation of MAPKs is associated with LPS-induced inflammation through iNOS expression and NF-κ activation.

Therefore, in the present invention, it was found that phosphorylation of intracellular signaling molecules (p-p38 MAPK, p-ERK (1/2), p-SAPK/JNK, p-IkBα and p-NF-kB) was significantly reduced. This means that up to about 25-30% of the controllable anti-inflammatory function occurs through multiple signal transduction pathways compared to cells induced by LPS inflammation by BGN(F) treatment. As another anti-inflammatory action, it was analyzed that the polarization of macrophages could outline a series of inflammation-related signaling events in inflammatory M1 macrophages activated by LPS upon interaction with BGN(F) (Fig. 8).

From the results of Examples 1 to 9, It was confirmed that BGN and folic acid-bound bioactive nanoparticles (BGN(F)) had anti-inflammatory properties under in vitro and in vivo conditions. Treatment of titer concentrations (80 and 160 μg/ml) BGN and BGN(F) increased the viability of LPS-treated inflammation-induced cells by decreasing cell death, LDH release and ROS production. Treatment with BGN(F) down-regulated the expression of pro-inflammatory molecules (TNF-α, IL-6, iNOS and COX-2) through inhibition of phosphorylation of molecules involved in intracellular signaling cascades. In addition, treatment with BGN and BGN(F) showed the potential to switch macrophage secretion from pro-inflammatory M1 to anti-inflammatory M2 states. The ionic products released from BGN and BGN(F) were found to mainly contribute to the anti-inflammatory function observed in vitro. As an in vivo experiment, injection of BGN and BGN(F) for toxin-induced muscle injury significantly lowered the expression of inflammatory signals (IL-6 and TNF-) and recruited macrophages around the damaged tissue to convert the phenotype to anti-inflammatory M2. did it Through the above examples, it was confirmed that BGN and BGN(F) can be used to control inflammatory diseases as a nanotherapy platform that does not use a separate drug.

The fact that the bioactive nanoparticles 'BGN(F)' itself possess the therapeutic function of an anti-inflammatory agent is informative to provide a new strategy as an application for alleviating the inflammatory response, and the unique nanoparticles can be applied to other inflamed tissues. analyzed to imply that there is

Claims (8)

A pharmaceutical composition for preventing or treating inflammatory diseases, comprising, as an active ingredient, ion-releasing mesoporous bioactive glass nanoparticles (BGn) containing silica (Si) and calcium (Ca). According to claim 1,
The ratio of the silica and calcium is 90:10 to 20:80 (molar ratio) will, a pharmaceutical composition for the prevention or treatment of inflammatory diseases. According to claim 1,
Wherein the mesoporous bioactive glass nanoparticles emit silica ions and calcium ions, a pharmaceutical composition for the prevention or treatment of inflammatory diseases. 4. The method of claim 3,
The amount of silica ions released is 150 to 300 ppm based on 50 mg of mesoporous bioactive glass nanoparticles, a pharmaceutical composition for preventing or treating inflammatory diseases. 4. The method of claim 3,
The amount of calcium ions released is 500 to 800 ppm based on 50 mg of mesoporous bioactive glass nanoparticles, a pharmaceutical composition for preventing or treating inflammatory diseases. According to claim 1,
The pharmaceutical composition for the prevention or treatment of inflammatory diseases, wherein the mesoporous bioactive glass nanoparticles are in a form in which folic acid is additionally bound to the surface thereof. According to claim 1,
The inflammatory diseases include erythematosus, atopy, rheumatoid arthritis, Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type 1 diabetes, lupus, chronic fatigue syndrome, fibromyalgia, hypothyroidism and hyperthyroidism, scleroderma, Behcet's disease, inflammatory bowel disease ( inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Meniere's syndrome, Guilian-Barre syndrome, Sjogren's syndrome, vitiligo, endometriosis, psoriasis, vitiligo or systemic scleroderma Phosphorus, a pharmaceutical composition for the prevention or treatment of inflammatory diseases. A method for preventing or treating an inflammatory disease, comprising administering the composition of any one of claims 1 to 7 to an individual other than a human having an inflammatory disease or fear of developing an inflammatory disease.

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