JP2013091612A - Immunosuppressant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunosuppressant that is suitable for treatment of diseases caused by an activated T cell and can be taken safely without anxiety for long terms.SOLUTION: The immunosuppressant comprises as an effective ingredient an alkylresorcinol represented by general formula (I). In general formula (I), Ris a saturated or unsaturated alkyl group; and Ris a hydrogen atom or a methyl group. In general formula (I), Ris preferably bonded to the para-position relative to R, and Ris preferably a 13-27C saturated or unsaturated alkyl group.

Description

  The present invention relates to T cells that cause diseases such as inflammatory reactions, autoimmune diseases, organ disorders, rejection at the time of organ transplantation / bone marrow transplantation, allergic diseases and the like caused by activated T cells with low anti-inflammatory drug effects The present invention relates to an immunosuppressive agent that can be prepared from a plant and suppresses activation.

  As an immunosuppressant, tacrolimus (FK506) and cyclosporin A are known to have an effective immunosuppressive effect. These immunosuppressants have been put to practical use in the medical field, but side effects such as cytopenia, nephrotoxicity and diabetes associated with long-term administration have been revealed.

  On the other hand, 4-alkylresorcinol is industrially used as a component of pharmaceuticals and cosmetics or an intermediate thereof, and as an additive or raw material of a resin. For example, 4-alkylresorcinol is useful in cosmetics and external skin products such as antibacterial action against acne-causing bacteria (Patent Document 1), antibacterial action against dandruff-causing bacteria (Patent Document 2), and whitening action (Patent Documents 3 to 4). 4-n-butylresorcinol has already been put into practical use as a whitening agent in cosmetics. Further, 4-n-hexylresorcinol is used as an anthelmintic agent for roundworms and duodenum. Furthermore, resorcinol derivatives are also known to have effective effects in pharmaceuticals, such as fat reducing action (Patent Document 5), anti-arteriosclerosis, anti-obesity and anti-diabetic actions (Patent Document 6) in obese patients.

  Alkylresorcinols have been reported to be widely contained in plants as resorcinol lipids, which are natural non-isoterpenoid phenolic amphiphilic compounds (Non-patent Document 1). In Non-Patent Document 1, as a plant serving as a source of resorcinol lipids, Urushiaceae, Ginkgoaceae, Porcupineaceae, Yabukouji, Primulaceae, Stigma, Iridaceae, Araceae, Asteraceae, Artemisia, Legume, Gramineae Has been reported. Regarding the toxicity of alkylresorcinols, even when 4-alkylresorcinol having a saturated alkyl group of 15 carbon atoms derived from cashew nut shell is orally administered to rats in an amount of 5 g / kg body weight, the obvious toxicity is It has been reported that this was not observed (Non-patent Document 2).

Japanese Patent No. 2875374 Japanese Patent No. 2801960 Japanese Patent Laid-Open No. 5-004905 Japanese Patent Publication No. 6-051619 Japanese Patent No. 2842041 JP-A-2005-068132

Chemical Reviews (1999), Vol.99, No.1, pp.1-25, Arkadiusz Kozubek et al. Nutrition Reviews (2004), Vol.62, No.3, pp.81-95, Alastair B. Ross et al.

  Numerous experiments and research have developed treatments for diseases caused by activated T cells. For example, for rheumatoid arthritis, systemic lupus erythematosus, and scleroderma, steroids and nonsteroidal anti-inflammatory drugs ( NSAIDs), immunosuppressants are used. However, steroids and NSAIDs are only treatments for the disease. In addition, various immunosuppressive agents are used to suppress rejection at the time of organ transplantation, but many have serious side effects such as cytopenia, renal failure, and shock symptoms.

  In addition to the above-mentioned diseases, inflammatory disorders that depend on activated T cells include asthma, allergy, psoriatic arthritis, endotoxia, type I diabetes, inflammatory bowel disease (IBD), colitis, multiple sclerosis (MS), transplant rejection, amyotrophic lateral sclerosis, demyelinating disorder, as well as various autoimmune diseases such as Crohn's disease, Sjogren's syndrome, and autoimmune hemolytic anemia. For these diseases, as described above, there is no active inhibitor for activated T cells that is safe, safe and suitable for treatment even after long-term use.

  Accordingly, an object of the present invention is to provide an immunosuppressive agent that is suitable for treatment of diseases caused by activated T cells and is safe and safe even after long-term use.

  As a result of intensive studies, the present inventors have found that alkylresorcinols known to be contained in wheat components have an action of suppressing the activity of activated T cells, and as a result, are used safely. It was possible to provide possible immunosuppressive agents.

  That is, this invention provides the immunosuppressant which uses the alkyl resorcinol represented by the following general formula (I) as an active ingredient.

（式中、Ｒ₁は飽和または不飽和のアルキル基を表し、Ｒ₂は水素原子またはメチル基を表す。）

(In the formula, R ₁ represents a saturated or unsaturated alkyl group, and R ₂ represents a hydrogen atom or a methyl group.)

  ADVANTAGE OF THE INVENTION According to this invention, the safe and safe immunosuppressive agent suitable for the treatment of the disease resulting from activated T cell can be provided even if it takes for a long period of time.

FIG. 1 is a graph showing the relationship between the dose of the immunosuppressive agent of Example 1 or 2 and the amount of various cytokine production in the measurement of cytokine production in Test Example 1-1, (a) is the amount of IL-2 production, (B) is IL-4 production, (c) is IFN-γ production, (d) is IL-6 production, (e) is IL-5 production, (f) is IL-10 production, (G) is a result about IL-12p40 production amount. FIG. 2 is a graph showing the relationship between the dose of the immunosuppressive agent of Example 1 or 2 and various cytokine production amounts in the measurement of cytokine production amount of Test Example 1-2, (a) is the IL-2 production amount, (B) IL-4 production, (c) IFN-γ production, (d) IL-6 production, (e) IL-5 production, (f) IL-10 production Is the result of FIG. 3 is a graph showing the relationship between the dose of the immunosuppressive agent of Example 1 or 2 and the absorbance in the Viability test (MTT assay) of the cells of Test Example 2.

  Hereinafter, the immunosuppressive agent of the present invention will be described in detail based on preferred embodiments.

  The immunosuppressive agent of the present invention comprises an alkylresorcinol represented by the above general formula (I) as an active ingredient. The alkylresorcinols have an action of suppressing T cell activation.

The saturated or unsaturated alkyl group represented by R ₁ in the general formula (I) is not limited by the number of carbon atoms, but more reliably exhibits an excellent activity suppressing action on activated T cells. From the viewpoint of making it, the number of carbon atoms is preferably 13 to 27.

  Typical examples of the saturated alkyl group having 13 to 27 carbon atoms include n-tridecyl, n-pentadecyl, n-heptadecyl, n-nonadecyl, n-henicosyl, n-tricosyl, n-pentacosyl and n-heptacosyl. A linear thing is mentioned, In addition to this, a branched or cyclic thing may be sufficient. Among these, a saturated alkyl group having 15 or 17 carbon atoms is preferable, and a linear saturated alkyl group having 15 or 17 carbon atoms is more preferable.

  Examples of the unsaturated alkyl group having 13 to 27 carbon atoms include those corresponding to the above saturated alkyl group having 13 to 27 carbon atoms. There is no restriction | limiting in particular in the number and position of the unsaturated bond contained in an unsaturated alkyl group.

In addition, from the viewpoint of activity-suppressing action on activated T cells, R ₂ in the above general formula (I) is preferably a hydrogen atom, and R ₁ is bonded to R ₂ in the para position. Is preferred.

Specific examples of the alkylresorcinols represented by the above general formula (I) used as an active ingredient in the immunosuppressive agent of the present invention include the following.
1,3-dihydroxy-5-n-tridecylbenzene (C13: 0)
1,3-dihydroxy-5-n-pentadecylbenzene (C15: 0)
1,3-dihydroxy-5-n-heptadecylbenzene (C17: 0)
1,3-dihydroxy-5-n-nonadecylbenzene (C19: 0)
1,3-dihydroxy-5-n-henicosylbenzene (C21: 0)
1,3-dihydroxy-5-n-tricosylbenzene (C23: 0)
1,3-dihydroxy-5-n-pentacosylbenzene (C25: 0)
1,3-dihydroxy-5-n-heptacosylbenzene (C27: 0)

As the alkylresorcinols represented by the above general formula (I), those in which R ₁ is a saturated alkyl group having 15 or 17 carbon atoms and R ₂ is a hydrogen atom are particularly preferable. Dihydroxy-5-n-pentadecylbenzene (C15: 0) and 1,3-dihydroxy-5-n-heptadecylbenzene (C17: 0) are preferred.

  The alkylresorcinols represented by the above general formula (I) can be obtained as commercial products, and those extracted from plants by a conventional method can also be used. Examples of plants containing alkylresorcinols include Urushiaceae, Ginkgoaceae, Porcupineaceae, Yabukouzi, Primulaceae, Nikuzukuidae, Iridaceae, Araceae, Artemisia, Leguminosae, Gramineae, and the like. Among these plants, gramineous plants have been studied for alkylresorcinols as edible active ingredients, and are suitable as a source of active ingredients used in the immunosuppressive agent of the present invention. Among grasses, wheat and rye are particularly suitable as a source because the content of alkylresorcinols represented by the above general formula (I) is as high as about 0.015 to 0.3% by mass of the whole grain weight. .

  The immunosuppressive agent of the present invention is an active ingredient, an alkylresorcinol represented by the above general formula (I), and various pharmaceutically acceptable carriers, excipients, stabilizers, etc. The additive and other components are contained. The immunosuppressive agent of the present invention can be formulated by a conventional method. In this case, the dosage form of the immunosuppressive agent of the present invention is an oral agent such as a tablet, powder, granule, capsule, or the above general formula. It is a parenteral agent such as an injectable preparation in which an alkylresorcinol represented by (I) is dissolved in sterilized distilled water or sterilized physiological saline together with a solubilizing agent and enclosed in an ampule. In addition, as other components, other components having medicinal effects, anti-inflammatory drugs, various vitamins, herbal medicines, and minerals can be appropriately blended.

  The content of the active ingredient in the immunosuppressive agent of the present invention is not particularly limited, and can be appropriately changed depending on the dosage form of the immunosuppressive agent, the symptom and age sex of the administered person, etc. When the target is used, it is usually preferable that the dose of the active ingredient of the immunosuppressive agent of the present invention is 0.01 to 10 g per day per adult.

  EXAMPLES Hereinafter, although an Example and a test example are given and this invention is demonstrated further in detail, this invention is not limited by these Examples and a test example.

  Below, DO11. Which is a transgenic mouse of a T cell receptor gene that recognizes 323-339 residues of chicken egg albumin (OVA) using two types of commercially available alkylresorcinols as immunosuppressants of Examples 1 and 2. Spleen cells of 10 mice, and T cells isolated from the spleen cells were added with an immunosuppressive agent to measure antigen stimulation response (cytokine production, antibody production) (Test Examples 1-1 and 1-2) and A Viability test (MTT assay; Test Example 2) was performed. In Test Example 1-1, the spleen cells were stimulated with OVA, which is a specific antigen, and the test was performed. In Test Example 1-2, the T cell receptor isolated from the spleen cells was treated with an anti-mouse CD3ε monoclonal antibody. The test was conducted with direct stimulation.

<Test mouse>
30-week-old female DO11.10 mice bred with SPF (bred in a SPF bred facility licensed from Jackson Laboratory)
<Test sample>
(1) Immunosuppressant Example 1; 1,3-dihydroxy-5-n-pentadecylbenzene (C15: 0)
Example 2; 1,3-dihydroxy-5-n-heptadecylbenzene (C17: 0)
All of the above compounds were purchased from ResearchChem GmbH (purity> 95% HPLC).
(2) Ovalbumin (OVA); Seikagaku Corporation, fraction V
(3) Anti-mouse CD3ε Armenian hamster monoclonal antibody; clone 145-2c11 (BD Pharmamingen, functional grade)

  In the following test, spleen cells collected from a plurality of mice were mixed, and then the test was first divided into three groups, and the test results were averaged.

[Test Example 1-1] Measurement 1 of cytokine production
Mixed spleen cells 3 × 10 ⁵ cells / well for two DO11.10 mice were used as test subjects. In a 96-well plate, RPMI (10% FCS) medium containing OVA (7.5 μM) and immunosuppressant (0 / 2.5 / 5/10 μM) at 5% CO ₂ , 37 ° C., total 300 μL / well Stimulated and cultured. At 48 hours after the start of the culture, the cell culture supernatant of the experimental group of cytokines IL-2, IL-4, and IL-5 was added to the cytokines IL-6, IL-10, IL-12p40, and IFN-γ at 72 hours. The cell culture supernatant of the experimental group was collected.
The concentration of cytokine (IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40, IFN-γ) in the collected cell culture supernatant was measured by ELISA. For the measurement of each cytokine, an analysis kit “anti-mouse cytokine ready-set-go!” Manufactured by eBioscience was used. The measurement was performed with duplicate. The measurement results are shown in FIG.

[Test Example 1-2] Measurement 2 of cytokine production
The day before the test, the anti-mouse CD3ε monoclonal antibody (clone 145-2c11, IgG concentration 0.5 mg / mL) was diluted with PBS to a concentration of 0.5 μg / mL, and 50 μL each was placed in a well and the plate was overnight at 4 ° C. Coated. Plates were washed 3 times with PBS on the day of testing.
Spleen-derived CD4 ⁺ T cells were separated using anti-mouse CD4 beads (Miltenyi. Biotec) using mixed spleen cells from two DO11.10 mice as materials. In a 96-well plate with 1 × 10 ⁵ cells / well of separated cells as test subjects, 5% CO ₂ , 37 ° C., total 300 μL / total in RPMI (10% FCS) medium containing an immunosuppressant (0/10 μM) Stimulated with well and cultured. At 48 hours after the start of the culture, the cell culture supernatant of the experimental group of cytokines IL-2, IL-4, and IL-5, and the cells of the experimental group of cytokines IL-6, IL-10, and IFN-γ at 72 hours. The culture supernatant was collected.
The concentration of cytokine (IL-2, IL-4, IL-5, IL-6, IL-10, IFN-γ) in the collected cell culture supernatant was measured by ELISA. For the measurement of each cytokine, an analysis kit “anti-mouse cytokine ready-set-go!” Manufactured by eBioscience was used. The measurement was performed with duplicate. The measurement results are shown in FIG.

[Test Example 2] Cell Viability Test (MTT Assay)
Mixed spleen cells 1 × 10 ⁵ cells / well for two DO11.10 mice were used as test subjects. The cells were cultured in a 96-well plate in RPMI (10% FCS) medium containing an immunosuppressant (0 / 2.5 / 5/10 μM) at 5% CO ₂ , 37 ° C., total 300 μL / well. Three days after the start of culture, 10 μL / well of MTT reagent (WST-1, Takara Bio) was added, and the absorbance at 450 nm was measured. The measurement was performed with duplicate. The measurement results are shown in FIG.

〔Test results〕
FIG. 1 shows the results of antigen-specific immune response suppression using DO11.10 mouse-derived spleen cells. FIG. 2 shows the results of suppression of a T cell-specific immune response using T cells obtained after isolating CD4 ⁺ T cells from spleen cells derived from DO11.10 mice. FIG. 3 shows the results of cytotoxicity of immunosuppressive agents against spleen cells derived from DO11.10 mice.

From FIG. 1, spleen cells of DO11.10 mice stimulated with OVA, a specific antigen, were cultured in a medium to which the immunosuppressant of the present invention was added. of CD4 ⁺ T IL-2 1 T-helper cell type in cells (Th1 cells) produce (FIG. 1 (a)) or IFN-gamma (Fig. 1 (c)), 2 activated CD4 ⁺ T cells IL-4 (FIG. 1 (b)), IL-5 (FIG. 1 (e)), IL-6 (FIG. 1 (d)), IL-10 (FIG. 1) produced by type T helper cells (Th2 cells) The production amount of (f)) was significantly suppressed in a dose-dependent manner with the immunosuppressant, indicating that the immune activity of activated Th1 and Th2 cells was suppressed. At the same time, the production amount of IL-12p40 (FIG. 1 (g)) that induces cell differentiation into Th1 was also significantly suppressed in a dose-dependent manner with the immunosuppressant.

As is clear from FIG. 2, in Test Example 1-2, as in Test Example 1-1, the production amount of various cytokines was significantly suppressed by the addition of an immunosuppressive agent. From this, the result of Test Example 1-1 originates from activated CD4 ⁺ T cells in spleen cells, and the immunosuppressive agent of the present invention directly suppresses the activity of activated CD4 ⁺ T cells. It was shown that.

  In Test Example 2, as apparent from FIG. 3, when the immunosuppressant of the present invention was added, the absorbance was not significantly different from the case of no immunosuppressant added, regardless of the dose. That is, in Test Example 2, an increase in spleen cell death due to the addition of an immunosuppressant could not be detected. From this, it was found that the immunosuppressive agent of the present invention is safe without showing cytotoxicity in the dose range of this test.

Claims (4)

An immunosuppressant comprising an alkylresorcinol represented by the following general formula (I) as an active ingredient.

(In the formula, R ₁ represents a saturated or unsaturated alkyl group, and R ₂ represents a hydrogen atom or a methyl group.) The immunosuppressant according to claim 1, wherein R ₁ in the general formula (I) is bonded to the para position with respect to R ₂ . The immunosuppressive agent according to claim 1 or 2, wherein R ₁ in the general formula (I) is a saturated or unsaturated alkyl group having 13 to 27 carbon atoms. The immunosuppressive agent according to claim 1 or 2, wherein R ₁ in the general formula (I) is a saturated alkyl group having 15 or 17 carbon atoms, and R ₂ is a hydrogen atom.

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