JP2006008717A - Interleukin inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new highly safe medicine for inhibiting interleukin, and further to provide a therapeutic agent for diseases, such as inflammatory diseases, allergic diseases, rheumatics, asthma, and multiple myeloma, in which interleukin is estimated to participate. <P>SOLUTION: The interleukin inhibitor and the therapeutic agent for diseases in which interleukin is estimated to participate, each contains astaxanthin and/or its ester as an active ingredient. The lipoxygenase inhibitor is useful for treating various diseases in which at least one interleukin selected from the group consisting of IL-4, IL-6 and IL-8 is estimated to participate. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an interleukin inhibitor. More specifically, the present invention relates to an inhibitor of at least one of interleukin-4, interleukin-6, and interleukin-8.

  Numerous cytokines have been discovered as protein factors that suppress the expression of biological functions such as immune responses, inflammatory reactions, and hematopoietic reactions in the living body. As its structure and action are elucidated, it is clear that the action of cytokines affects not only the immune system but also various functions of the living body, and is closely related to the development, differentiation, homeostasis, pathophysiology, etc. of the living body It has become.

  Helper T cells (Th) that play a central role in the immune response are classified into Th1 cells and Th2 cells (Non-patent Document 1). Th2 cells produce cytokines such as interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-13 (IL-13).

  Among these, IL-4, like IL-13, induces IgE antibody production by acting on B cells and inducing a class switch to IgE. The produced IgE antibody binds to the surface of mast cells, and when a specific antigen binds to this IgE antibody, mast cells (mast cells) are activated. From the activated mast cells, inflammatory substances such as histamine stored in the cells are released by de-aggregation, and allergic symptoms are induced. Furthermore, IL-4 also has the ability to differentiate and proliferate from naive T cells that have not been antigen-sensitized to Th2 cells and to activate mast cells.

  IL-4 induces IgE antibody production as well as mast cell activation and proliferation. It also induces gene expression of VCAM-1, which is an important molecule that functions when eosinophils adhere to vascular endothelial cells and infiltrate tissues. Furthermore, IL-4 acts on naive T cells, which are progenitor cells of helper T cells, induces functional differentiation to Th2, and also functions as a growth factor for T cells after differentiation and maturation. Furthermore, Th2 is not only an immediate allergic reaction involving IgE antibodies and mast cells but also a central cell that induces a delayed allergic reaction involving eosinophils. IL-4 plays an important role as a differentiation and growth factor for Th2, while it is produced from Th2 and is an important cytokine deeply involved in both immediate and delayed allergic reactions. Examples of such diseases related to abnormal production or secretion of IL-4 include allergic diseases, parasitic infections, autoimmune diseases such as systemic lupus erythematosus, viral or bacterial infections, malignant tumors, HVG (Host-versusus -Graft) disease or acquired immunodeficiency syndrome (AIDS).

  In order for IL-4 to exhibit biological activity, information must be transmitted into the cell after binding to a specific receptor on the target cell. With the recent development of molecular biology, the intracellular signal transduction mechanism from the IL-4 receptor has been elucidated, and major intracellular molecular groups have been identified. Among them, Stat 6 has been found as a particularly important molecule (Non-patent Document 2). Therefore, stat 6 activation inhibitors have been studied as inhibitors of IL-4 (for example, Patent Document 1).

  Interleukin-6 (IL-6) is known as a cytokine that induces terminal differentiation of B cells into antibody-producing cells. IL-6 not only plays an important role in the antibody production system of B cells, but also induces proliferation and differentiation in T cells, acts on hepatocytes to induce synthesis of acute phase proteins, hematopoiesis It has been clarified that it is an important factor not only in the immune system but also in the body defense system such as the hematopoietic system, the nervous system, and the liver.

  Diseases associated with abnormal production or secretion of IL-6 include cancer cachexia, atrial myxoma, rheumatoid arthritis, autoimmune disease, Castleman's disease, myeloma, Rennert lymphoma, mesangial proliferative nephritis, psoriasis And Kaposi sarcoma associated with AIDS, postmenopausal osteoporosis, and the like. Various studies on IL-6 inhibitors have been conducted for the purpose of treatment and prevention of these diseases (for example, Patent Documents 2 to 6).

  IL-8 is a chemotactic factor for neutrophils, T cells, and basophils that activates neutrophils and releases lysosomal enzymes, altering neutrophil adhesion to vascular endothelial cells, Moreover, Candida growth suppression is enhanced. In addition, when injected into the joint, the joint synovium is destroyed with a large amount of neutrophil wetness. Furthermore, the expression of adhesion factor on neutrophils is enhanced, histamine release is regulated by basophils, It has many physiological activities such as neutrophil activation. IL-8 is also called an inflammatory cytokine, and abnormal occurrence of IL-8 and excessive reaction to IL-8 are considered to cause inflammatory diseases.

  Inflammatory diseases involving IL-8 include inflammatory skin diseases such as inflammatory keratosis (psoriasis, etc.), atopic dermatitis, contact dermatitis; rheumatoid arthritis, systemic lupus erythematosus (SLE), Behcet Autoimmune diseases that are chronic inflammatory diseases such as illness; inflammatory bowel diseases such as Crohn's disease and ulcerative colitis; inflammatory liver diseases such as hepatitis B, hepatitis C, alcoholic hepatitis, drug allergic hepatitis; Inflammatory kidney diseases such as glomerulonephritis; inflammatory respiratory diseases such as bronchitis; stomatitis, vocal fold inflammation, inflammation that occurs when using artificial organs and blood vessels. Therefore, various IL-8 inhibitors have been examined (for example, Patent Documents 5 to 9).

As described above, various interleukin inhibitors have been studied. Although there are inhibitors that inhibit both IL-6 and IL-8 (for example, Patent Documents 5 and 6), IL-4 is also included. There are no known inhibitors for a wide range of interleukins.
JP 2000-229959 A JP 2005-22972 A JP-A-8-169840 Japanese Patent Laid-Open No. 7-324097 JP-A-8-73453 International Publication No. 2002/049632 Pamphlet JP 2004-269450 A Japanese Patent Laid-Open No. 11-158071 Special Table 2000-507556 Mosmann TR et al., J. Immunol., 1986, 136, pp. 2348-2357 McKnight, Science, 1994, 265, pp.1701-1706

  An object of this invention is to provide the new highly safe chemical | medical agent which has an inhibitory action with respect to various interleukins.

  The present invention provides an interleukin inhibitor containing astaxanthin and / or an ester thereof as an active ingredient, wherein the interleukin is selected from the group consisting of interleukin-4, interleukin-6, and interleukin-8. The

  The present invention also provides a therapeutic agent for a disease state involving at least one of interleukin-4, interleukin-6, and interleukin-8, which contains astaxanthin and / or an ester thereof as an active ingredient.

  In a preferred embodiment, the condition is inflammatory disease, allergic disease, rheumatism, asthma, or multiple myeloma.

  According to the present invention, a new interleukin inhibitor is provided. This interleukin inhibitor is an inflammatory disease, allergic disease, rheumatism, asthma, multiple disease that is considered to involve at least one interleukin selected from the group consisting of IL-4, IL-6, and IL-8. It can be used as a therapeutic agent for various diseases such as myeloma. Since the interleukin inhibitor of the present invention has low toxicity, it has high safety.

  Astaxanthin and / or its ester, which is an active ingredient of the interleukin inhibitor of the present invention, has the following formula:

(Here, R ¹ and R ² are each independently a hydrogen atom or a fatty acid residue). The ester of astaxanthin is not particularly limited. For example, saturated fatty acids such as palmitic acid and stearic acid, or oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, bishomo-γ-linolenic acid, arachidonic acid, Examples thereof include monoesters or diesters of unsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid. These can be used alone or in appropriate combination. Astaxanthin has a structure having extra oxo groups and hydroxy groups at both ends of the skeleton of β-carotene, and therefore has low molecular stability unlike β-carotene. On the other hand, the ester body (for example, krill extract) in which the hydroxyl groups at both ends are esterified with unsaturated fatty acid or the like is more stable.

  Astaxanthin and / or its ester used in the present invention may be either chemically synthesized or derived from natural products. Examples of the latter natural products include red yeast containing astaxanthin and / or an ester thereof; shellfish shells such as tigliopath (red daphnia) and krill; and microalgae such as green algae. In the present invention, an extract containing astaxanthin and / or its ester produced by any method can be used as long as the characteristics of astaxanthin and / or its ester can be utilized. In general, extracts from these natural products are used, and the extract may be in the form of an extract or may be appropriately purified as necessary. In the present invention, such a crude extract or crushed powder containing astaxanthin and / or an ester thereof, or an appropriately purified or chemically synthesized product containing astaxanthin and / or its ester may be used alone or in appropriate combination. Can do. Considering the stability in the body, an ester form is preferably used.

  The interleukin inhibitors of the present invention may be useful for treating or preventing a disease or condition involving at least one interleukin selected from the group consisting of IL-4, IL-6, and IL-8. . Such diseases or symptoms include allergic diseases, parasitic infections, autoimmune diseases such as systemic lupus erythematosus, viral or bacterial infections, malignant tumors (plasmacytoma, multiple myeloma, cancer cachexia, Atrial myxoma, myeloma, Rennert lymphoma, etc.), HVG (Host-versus-Graft) disease or acquired immune deficiency syndrome (AIDS), autoimmune disease (rheumatoid arthritis, systemic lupus erythematosus (SLE), Behcet's disease, etc. ), Castleman's disease, Kaposi sarcoma associated with AIDS, postmenopausal osteoporosis, inflammatory skin diseases (such as inflammatory keratosis, atopic dermatitis, contact dermatitis), inflammatory bowel disease (Crohn's disease, ulcerative) Colitis, etc.), inflammatory liver diseases (hepatitis B, hepatitis C, alcoholic hepatitis, drug allergic hepatitis, etc.), inflammation Sex kidney disease (such as glomerulonephritis), inflammatory respiratory disease (asthma, chronic obstructive pulmonary disease, bronchitis, etc.), stomatitis, vocal cord inflammation, such as inflammation and the like that occurs when artificial organs and artificial blood vessel used.

  The therapeutic agent for diseases involving interleukins of the present invention contains astaxanthin and / or an ester thereof as an active ingredient, similar to the above-described interleukin inhibitors of the present invention. It is particularly useful for the treatment of inflammatory diseases, allergic diseases, rheumatism, asthma, or multiple myeloma.

  The administration route of the interleukin inhibitor of the present invention or the therapeutic agent for a disease involving at least one interleukin selected from the group consisting of IL-4, IL-6, and IL-8 is oral administration or parenteral administration Any of these may be used. The dosage form is appropriately selected depending on the administration route. For example, injections, infusions, powders, granules, tablets, capsules, pills, enteric solvents, troches, liquids for internal use, suspensions, emulsions, syrups, liquids for external use, poultices, nasal drops, ear drops Agents, eye drops, inhalants, ointments, lotions, suppositories, enteral nutrients and the like. This can be used alone or in combination depending on the symptoms. In these preparations, auxiliary agents usually used in the pharmaceutical preparation technical field such as excipients, binders, preservatives, oxidation stabilizers, disintegrants, lubricants, and corrigents are used as necessary. .

  The dose of the interleukin inhibitor of the present invention or the therapeutic agent for a disease associated with interleukin varies depending on the purpose of administration and the situation (gender, age, weight, etc.) of the administration subject. Usually, for adults, 0.1 mg to 2 g per day, preferably 4 mg to 500 mg per day in the case of oral administration, in terms of astaxanthin free body, while 0.01 mg to 1 g per day for parenteral administration, Preferably it can be administered at 0.1 mg to 500 mg.

  The interleukin inhibitors of the present invention can be used not only as pharmaceuticals as described above, but also as quasi drugs, cosmetics, functional foods, nutritional supplements, foods and drinks, and the like. When used as a quasi-drug or cosmetic, it can be used together with various adjuvants commonly used in the technical field such as quasi-drug or cosmetic, if necessary. Or, when used as a functional food, nutritional supplement, or food and drink, it is usually used in foods such as sweeteners, spices, seasonings, preservatives, preservatives, bactericides, and antioxidants as necessary. You may use with the additive used. Further, it may be used in a desired shape such as solution, suspension, syrup, granule, cream, paste, jelly, etc., or as necessary. The ratio contained in these is not specifically limited, It can select suitably according to a use purpose, a usage form, and a usage-amount.

(Preparation Example 1: Preparation of astaxanthin monoester)
Astaxanthin monoester was prepared as follows. The Haematococcus pulvialis strain K0084 was cultured at 25 ° C. under irradiating gas containing 3% CO ₂ under light irradiation conditions, under nutrient stress (nitrogen source deficiency), and cysted. The cysted cells were crushed by means commonly used by those skilled in the art, and the oily fraction was extracted with ethanol. The extract contained lipids such as triglycerides in addition to astaxanthins. The extract was subjected to column chromatography using a synthetic resin adsorbent to obtain a purified product containing an astaxanthin monoester. This purified product was analyzed by HPLC, and it was confirmed that this purified astaxanthin monoester contained a monoester having a molecular weight of 858 as a main component, did not contain free and diester forms of astaxanthin, and contained a slight amount of diglyceride. .

(Example 1: Effect on IL-4 release)
The effect of the astaxanthin monoester obtained in Preparation Example 1 on the release of IL-4 from the cells was examined. RPMI-1640, 10% FBS containing human peripheral blood mononuclear cells as cells releasing IL-4, and 100 U / mL penicillin, 100 μg / mL streptomycin, and 2 mM L-glutamine as incubation buffer (PH 7.4) was used.

  Human peripheral blood mononuclear cells were suspended in incubation buffer at 37 ° C., and concanavalin A was added as an IL-4 releasing factor. There, astaxanthin monoester dissolved in dimethyl sulfoxide (DMSO) was added to 250, 25, 2.5, 0.25, and 0.025 μM, and incubated at 37 ° C. for 16 hours. The DMSO concentration was 0.1 (v / v)%. Moreover, only DMSO was added to the negative control.

After completion of the incubation, the supernatant was collected by centrifugation, and the IL-4 concentration in the supernatant was measured by ELISA. The 50% inhibitory concentration (IC ₅₀ ) by astaxanthin monoester was determined when the IL-4 concentration in the negative control was 100%. As a result, IC ₅₀ was 74.1 μM. The inhibition rate of release at 250 μM was 99%. Thus, astaxanthin monoester showed a relatively low inhibitory concentration and a high inhibition rate for the release of IL-4.

(Example 2: Effect on IL-6 release)
The effect of the astaxanthin monoester obtained in Preparation Example 1 on the release of IL-6 from the cells was examined. Human peripheral blood mononuclear cells were used as cells releasing IL-6, and AIM-V medium (pH 7.4) was used as an incubation buffer.

  Human peripheral blood mononuclear cells were suspended in incubation buffer at 37 ° C., and lipopolysaccharide (LPS) was added as an IL-6 releasing factor. There, astaxanthin monoester dissolved in dimethyl sulfoxide (DMSO) was added to 250, 25, 2.5, 0.25, and 0.025 μM, and incubated at 37 ° C. for 16 hours. The DMSO concentration was 0.1 (v / v)%. Moreover, only DMSO was added to the negative control.

After completion of the incubation, the supernatant was collected by centrifugation, and the IL-6 concentration in the supernatant was measured by ELISA. The 50% inhibitory concentration (IC ₅₀ ) by astaxanthin monoester was determined when the IL-6 concentration in the negative control was 100%. As a result, the IC ₅₀ was 27.2 μM. The inhibition rate of release at 250 μM was 100%. Thus, astaxanthin monoester showed a relatively low inhibitory concentration and a high inhibition rate for the release of IL-6.

(Example 3: Effect on IL-8 release)
The release of IL-8 from cells was examined in the same manner as in Example 2 except that an anti-IL-8 antibody was used in place of the anti-IL-6 antibody in ELISA. As a result, the IC ₅₀ was 39.4 μM. The inhibition rate of release at 250 μM was 100%. Thus, astaxanthin monoester showed a relatively low inhibitory concentration and a high inhibition rate for the release of IL-8.

(Reference Example 1: Measurement of 50% lethal concentration against HUVEC)
Human umbilical vein endothelial cells (HUVEC) (ATCC CRL-1730) were obtained from American Type Culture Collection and endothelial cell growth containing 10% fetal bovine serum supplemented with 1% Antibiotic-Antimycotic (GIBCO BRL, USA) Medium (CELL APPLICATIONS, USA)) was pre-cultured at 37 ° C. in a 5% CO ₂ atmosphere.

  Matrigel matrix (BD Biosciences, USA) was thawed and kept on ice at 4 ° C., and 50 μL of matrix was transferred to each well of a 96-well tissue culture plate. The plate was incubated at 37 ° C. for at least 1 hour to solidify the matrix solution.

  On the other hand, the astaxanthin monoester obtained in Preparation Example 1 was dissolved in DMSO and then diluted with distilled water to obtain 25000, 2500, 250, 25, and 2.5 μM astaxanthin in 40 (v / v)% DMSO. A stock test solution containing the monoester was prepared.

100 μL of HUVEC suspension (approximately 2.5 × 10 ³ cells / well) was placed in a 96-well Matrigel plate at 37 ° C. in a 5% CO ₂ atmosphere. After 24 hours, 100 μL of growth medium and 2 μL of each of the above stock test solutions or vehicles (40 (v / v)% DMSO) were added to each of the two wells and incubated for a further 72 hours. The final concentrations of DMSO and astaxanthin monoester were 250, 25, 2.5, 0.25, and 0.025 μM.

  At the end of the incubation, 20 μL of 90% alamarBlue reagent was added to each well and incubated for an additional 6 hours. Next, the fluorescence intensity of each well was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm using a Spectrafluor Plus plate reader, and the number of viable cells was counted. This is based on the ability of viable cells to change alamarBlue from a non-fluorescent oxidized form (blue) to a fluorescent reduced form (red). The 50% lethal concentration was calculated as the concentration at which 50% of the number of cells at the start of the experiment.

As a result, the 50% lethal concentration (LC ₅₀ ) of astaxanthin monoester with respect to HUVEC was 250 μM (maximum dissolution concentration in DMSO), indicating that the toxicity was low.

  According to the present invention, a new interleukin inhibitor is provided. This interleukin inhibitor is an inflammatory disease, allergic disease, rheumatism, asthma, multiple disease that is considered to involve at least one interleukin selected from the group consisting of IL-4, IL-6, and IL-8. It can be used as a therapeutic agent for various diseases such as myeloma. Astaxanthin and / or its ester, which is an active ingredient of the interleukin inhibitor of the present invention, has a long diet and low toxicity, so it is extremely safe. Therefore, it can be used not only as a medicine but also on a daily basis as a health food.

Claims (3)

  An interleukin inhibitor containing astaxanthin and / or an ester thereof as an active ingredient, wherein the interleukin is selected from the group consisting of interleukin-4, interleukin-6, and interleukin-8 Inhibitor.   A therapeutic agent for a disease state involving at least one of interleukin-4, interleukin-6, and interleukin-8, comprising astaxanthin and / or an ester thereof as an active ingredient.   The therapeutic agent according to claim 2, wherein the pathological condition is inflammatory disease, allergic disease, rheumatism, asthma, or multiple myeloma.