JP5603457B2 - MIF secretion inhibitor - Google Patents

Description

  The present invention relates to a macrophage migration inhibitory factor (MIF) secretion inhibitor and an inflammatory disease ameliorating agent.

Macrophage migration inhibitory factor (hereinafter referred to as MIF) is one of the mediators of inflammation and immunity, and is a humoral factor that controls macrophage migration, collects macrophages at the site of inflammation, and enhances phagocytic ability. MIF is rheumatoid arthritis (Non-patent document 1: Expert Opin Ther Targets, 7, 2, 153-164, 2003), nephritis (Non-patent document 2: Expert Opin Ther Targets, 7, 2, 153-164, 2003), Atopic dermatitis (Non-patent document 3: Biochem Biophys Res Commun, 240, 11, 173-8, 1997), psoriasis (Non-patent document 4: BJ Dermatol, 141, 1061-66, 1999), ulcerative colitis, Sepsis, contact dermatitis (Non-patent document 5: Eur J Immunol, 33, 1478-87, 2003), inflammation caused by ultraviolet rays (Non-patent document 6: J Invest Dermatol, 112, 2, 210-15, 1999), delayed Acute / chronic inflammatory diseases (J End, 179, 15-23, 2003) such as sexual allergy (Non-patent Document 7: Pro Natl Acad Sci USA, 93, 7849-54, 1996), arthritis, graft rejection, tumor It is involved in the pathological processes of inflammatory diseases such as growth, angiogenesis, anemia, encephalomyelitis and the like, and it is expected to reduce these inflammatory diseases by inhibiting MIF.
Various active inhibitors of MIF have been developed (Patent Document 1: JP-T-2003-513065, Patent Document 2: JP-T-2005-500266, Patent Document 3: JP-T-2006-517777), MIF secretion inhibitors are not known.
The present inventor has focused on MIF secretion suppression and has continued research and development on MIF secretion inhibitors. However, the present inventor has previously proposed a technique that uses Himejoon or its extract as a MIF secretion inhibitor. Proposed in Japanese Patent Application No. 2008-022588.
Convolvulus is traditionally known as a tonic. (Non-Patent Document 8: Revised primary color Makino plant encyclopedia (joint flower / divided flower) edited by Tomita Makino, p 289). However, the secretory effect of MIF secretion by the convolvulus extract is not known.

Japanese translation of PCT publication No. 2003-513065 Japanese translation of PCT publication No. 2005-500026 JP-T-2006-517997 Expert Opin Ther Targets, 7, 2, 153-164,2003 Expert Opin Ther Targets, 7, 2, 153-164,2003 Biochem Biophys res Commun, 240, 11,173-8, 1997 B J Dermatol, 141, 1061-66, 1999 Eur J Immunol, 33, 1478-87, 2003 J Invest Dermatol, 112, 2, 210-15, 1999 Pro Natl Acad Sci USA, 93, 7849-54, 1996 Revised primary color Makino Plant Encyclopedia (Joint Ventures / Left Flower) edited by Tomita Makino, p 289

  An object of the present invention is to provide a MIF secretion inhibitor.

The present invention was completed by finding that the extract of the convolvulus (Calystegia japonica) has a macrophage migration inhibitory factor secretion inhibitory action.
The main configuration of the present invention is as follows.
1. Macrophage migration inhibitory factor secretion inhibitor containing an extract of convolvulus.
2.1. A skin external preparation comprising the described macrophage migration inhibitory factor secretion inhibitor.
3.1. An ameliorating agent for inflammatory diseases comprising the described macrophage migration inhibitory factor secretion inhibitor.

According to the present invention, it is possible to provide a new therapeutic agent or preventive agent for atopic dermatitis in which abnormal secretion of MIF is observed.
Abnormal secretion and involvement of MIF other than atopic dermatitis, skin inflammation caused by psoriasis and ultraviolet rays, hemangioma, urticaria, sepsis, respiratory inflammation, ulcerative colitis, rheumatoid arthritis, nephritis, contact It is also expected to be effective in treating and preventing acute and chronic inflammatory diseases such as dermatitis and delayed allergy, arthritis, graft rejection, tumor growth, angiogenesis, anemia, encephalomyelitis, and other inflammation caused by bacterial infection. By locally prescribing the MIF secretion inhibitor as an external preparation for skin, the function of macrophage migration inhibitory factor can be suppressed, and the function of macrophages in the inflammatory site of skin can be suppressed.

Table 1 is a graph of Table 1 showing the results of a UVB-induced MIF secretion inhibition test using keratinocytes. Table 2 is a graph of the results of LPS-induced MIF secretion inhibition test using THP-1 cells.

  Convolvulaceae (daytime) (scientific name Calystegia japonica) used in the present invention is a plant belonging to the genus Convolvulaceae and is a perennial plant distributed in Japan and China. A vine plant that blooms in summer.

As a convolvulus extract, convolvulus whole grass or flowers, stems, leaves, roots are crushed as they are, or dried and then crushed, water or alcohol such as ethanol, ether, acetone, 1,3-butylene glycol, A crude extract extracted with an organic solvent such as 1,2-pentanediol, propylene glycol, and ethyl acetate, and an extract obtained by stepwise purification of the crude extract with various types of chromatography such as partition extraction and column chromatography Includes product fractions. These may be used alone or in combination of two or more. Convolvulus whole grass or flowers, stems, leaves, and roots may be subjected to an extraction operation, but it is more efficient to perform extraction after processing such as shredding, drying, and pulverization. Extraction can be performed by immersing in an extraction solvent. In order to increase the extraction efficiency, the extraction solvent can be stirred, crushed and homogenized in the extraction solvent, or pressure can be applied in the extraction solvent. The extraction temperature is suitably about 5 to 100 ° C., and the extraction time is about 5 minutes to 1 month. These conditions can be set as appropriate.
The convolvulus extract can be used as it is, or in a state in which it is dissolved in water, an organic solvent such as ethanol, as a MIF secretion inhibitor, an inflammatory disease improving agent, particularly a skin inflammatory disease improving agent. Further, if necessary, the extraction solvent may be distilled off and the dried product may be used.

Convolvulus extract can be applied as a dry product in the range of 0.0001 to 1000 mg / day, and the amount thereof can be appropriately set according to the subject, application form, and symptoms, not limited to this range.
The blending amount of the convolvulus extract of the present invention is preferably about 0.0001 to 10% by weight, but the blending amount is appropriately set in a wide range up to 100% by weight depending on various conditions such as the dosage form to be used and the intended use. it can.

The preparation of the present invention can be administered orally or parenterally.
The preparation of the present invention can be applied in the form of, for example, a solution such as an aqueous solution, an oil, an emulsion, and a liquid, a semi-solid such as a gel and a cream, and a solid such as a powder, granule, capsule, microcapsule, and solid. . It is prepared in these forms by conventionally known methods, and lotions, emulsions, gels, creams, ointments, plasters, haps, aerosols, suppositories, injections, powders, granules, tablets, pills , Various dosage forms such as syrups and lozenges. These can be applied to the body by applying, sticking, spraying, drinking and the like. Among these dosage forms, lotions, emulsions, creams, ointments, plasters, haps, aerosols and the like, which are external preparations for skin, are suitable.
Usually, these dosage forms and compositions can be produced according to the formulation method used in medicine.
Hereinafter, the present invention will be described in detail.

Preparation of Convolvulus Extract 100 g of dried convolvulus whole grass was immersed in 6 L of 99.5% ethanol for 1 week, and the solvent of the obtained ethanol extract was distilled off to obtain 1 g of Convolvulus extract.

Bindweed extract MIF secretion inhibition test UVB-induced MIF secretion inhibition test using keratinocytes It is known that MIF is strongly expressed in keratinocytes, one of the constituent cells of the skin. It is known that MIF secretion is promoted by irradiating UVB to keratinocytes (J Invest Dermatol, 112, 2, 210-15, 1999). Therefore, it was examined whether or not the convolvulus extract suppresses the secretion of MIF when keratinocytes are irradiated with UVB.
Human keratinocytes were seeded in a φ35 mm dish at 7.0 × 104 cells / cm 2 and cultured in KBM / KGM medium (manufactured by LONZA) for 5 days. Change to KBM / KGM medium (BPE (-)) containing convolvulus extract, and after overnight culture, UVB is irradiated at 1.0 W / m2 and 20 mJ / m2 in the presence of HANKS (-). The culture medium was replaced with KBM / KGM medium (BPE (-)) containing, and cultured for 6 hours. The culture supernatant was collected, and the amount of MIF in the supernatant was measured using the hMIF ELISA kit according to a conventional method, and the amount of MIF secretion was calculated. Furthermore, the cells were lysed with Cell Lysis Buffer, the protein in the lysate was measured with a protein assay kit, and the amount of protein per dish was calculated. Concentrations of MIF per unit protein were compared when convolvulus extract was not added and UVB (-) treatment was 100%. The results are shown in Table 1. A graph of Table 1 is shown in FIG. In addition, as a result of comparing the protein amount of the cells to which the control and the convolvulus extract were added in this test, no difference was observed in the protein amount between the two, and the cytotoxicity of the convolvulus was not observed in this test system.

  By adding the convolvulus extract to keratinocytes, the secretion of MIF was remarkably suppressed. Expected to improve skin inflammatory diseases such as UV-induced skin damage / inflammation, atopic dermatitis, psoriasis, contact dermatitis, etc. .

2. LPS-induced MIF secretion inhibition test using THP-1 cells It is known that MIF secretion from macrophages is promoted by bacterial infection or stimulation of foreign substances. THP-1 cells, which are human monocytic leukemia cells, which are macrophage model cells, secrete MIF upon stimulation with LPS in a bacterial infection model (FEBS Let 551, 78-88, 2003). Therefore, it was investigated whether the convolvulus extract suppressed MIF secretion of THP-1 cells stimulated with LPS.

  THP-1 cells were cultured in 1% serum / RPMI medium for 3 hours, seeded at 1 × 10 6 cells / well (12 well plate) in the same medium, and convolvulus extract was added. 45 minutes after adding the convolvulus extract, LPS was added at a final concentration of 10 μg / ml and cultured for 4 hours. After culture, the cells were centrifuged at 400 × g, the cell supernatant was collected, the amount of MIF in the supernatant was measured according to a conventional method using an hMIF ELISA kit, and the amount of MIF secretion was calculated. Conservation of MIF was compared when convolvulus extract was added, with no convolvulus extract added and LPS (-) treatment 100%. The results are shown in Table 2. A graph of Table 2 is shown in FIG.

  By adding the convolvulus extract to THP-1 cells, secretion of MIF was suppressed. Prevention and improvement of inflammation caused by bacterial infection such as acne and sepsis, and chronic inflammatory diseases such as invasive inflammation, allergies and rheumatoid arthritis can be expected.

Claims (1)

A skin external preparation for inhibiting macrophage migration inhibitory factor secretion comprising a macrophage migration inhibitory factor secretion inhibitor comprising a convolvulus ethanol extract as an active ingredient .

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