JP2006282639A - Prostaglandin production inhibitor and anti-inflammatory preparation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new prostaglandin production inhibitor, and an anti-inflammatory preparation containing the same as an active ingredient. <P>SOLUTION: This prostaglandin production inhibitor is selected from chalcone compounds. The prostaglandin production inhibitor consists of licochalcone A or licochalcone B. The anti-inflammatory preparation is provided by containing the prostaglandin production inhibitor as the active ingredient. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a novel prostaglandin production inhibitor and an anti-inflammatory agent comprising the prostaglandin production inhibitor as an active ingredient.

IL-1β is one of cytokines that are expressed in the body during an immune response. IL-1β is mainly produced by monocytes, macrophages and their related cells when stimulated by endotoxin (LPS) and viruses, and prostaglandin E _{2 in} tissues such as hypothalamic thermoregulatory centers and vascular endothelial cells. (Hereinafter abbreviated as PGE ₂ ) causes production enhancement and osteoclast activation. As a result, fever, tissue damage, inflammatory reaction such as enhancement of pain caused by bradykinin, and promotion of bone resorption are caused. Therefore, IL-1β has been suggested to be involved in diseases such as rheumatoid arthritis and osteoporosis (for example, see Non-Patent Documents 1 and 2).

Therefore, a substance that inhibits PGE ₂ production by IL-1β may be effective as a pharmaceutical material for preventing and treating inflammatory reactions and diseases that occur in association with PGE _2, and is a novel IL-1β-inducing property. PGE ₂ production inhibitors are being sought. The present inventors have discovered a substance that inhibits PGE ₂ production by IL-l [beta] in the licorice extract were identified this. This material was lycochalcone A, a chalcone compound.

Lycochalcone A is a well-known compound and has been proposed for use as an antitumor agent, an antibacterial agent effective against methicillin-resistant Staphylococcus aureus, cosmetics, sebum secretion inhibitor, HIV gene expression inhibitor, and the like ( For example, refer nonpatent literatures 3 and 4 and patent documents 1-5). However, as described later, it has not been known so far that the compound has an IL-1β-induced PGE ₂ production inhibitory activity, which is a novel matter.
Ueno Yasunori, Oishi Sachiko, "Inflammation / Allergy and Prostanoids (1) Inflammation", Pharmacology of Japan (Folia Pharmacol. Jpn.), 117, 255-261 (2001) Yoichiro Iwakura, “Role of IL-1 in the Onset of Arthritis”, Latest Medicine, Vol. 57, No. 4, 16 (840) -24 (848), 2002.4 Tetrahedron Letters No.50, pp4461-4462,1975 Shoji Shibata et al., “Inhibitory Effects of Licochalcone A Isolated from Glycyrrhiza inflata Root on Inflammatory Ear Edema and Tumour Promotion in Mice”, Planta med. 57 (1991), pp221-224 JP-A-6-122623 JP-A-7-2656 JP-A-10-77221 JP 2001-163718 A JP 2001-253823 A

As described above, an IL-1β-inducible PGE ₂ production inhibitor is expected as a seed compound for a novel pharmaceutical product.
An object of the present invention is to provide a novel substance exhibiting an anti-inflammatory action by suppressing PGE ₂ production by IL-1β.

In order to achieve the above object, the present invention provides a prostaglandin production inhibitor selected from chalcone compounds.
The present invention also provides a prostaglandin production inhibitor consisting of lycochalcone A or lycochalcone B.
Furthermore, the present invention provides an anti-inflammatory agent comprising the prostaglandin production inhibitor as an active ingredient.

  ADVANTAGE OF THE INVENTION According to this invention, the novel prostaglandin production inhibitor and the anti-inflammatory agent which contains it as an active ingredient can be provided.

  The prostaglandin production inhibitor of the present invention comprises a chalcone compound having prostaglandin production inhibitory activity. In the present invention, a suitable chalcone compound is lycochalcone A or lycochalcone B, more preferably lycochalcone A represented by the following formula (1).

  Moreover, the prostaglandin production inhibitor of the present invention can form a pharmacologically acceptable salt with hydrochloric acid, phosphoric acid, organic acid, metal ion and the like. The prostaglandin production inhibitor of the present invention also includes a compound in the form of such a pharmacologically acceptable salt.

  Licochalcone A and Licochalcone B are well known. For example, Non-Patent Document 3 (Tetrahedron Letters No. 50, pp4461-4462,1975) describes the structures of Licochalcone A and Lycochalcone B. Non-Patent Document 4 (Shoji Shibata et al., “Inhibitory Effects of Licochalcone A Isolated from Glycyrrhiza inflata Root on Inflammatory Ear Edema and Tumor Promotion in Mice”, Planta med. 57 (1991), pp221-224) It describes the inhibitory effect of mouse ear edema and tumor formation. Non-patent document 4 describes that lycochalcone A is obtained by purifying an ethyl acetate extract of licorice (Xin-jiang liquorice) by silica gel column chromatography. Therefore, the compound can be obtained from commercially available licorice or the like by a known extraction method.

The prostaglandin production inhibitor of the present invention has PGE ₂ production inhibitory activity induced by IL-1β, as demonstrated in the Examples described later. IL-1β is one of the cytokines that are expressed during an immune response in a living body and is a substance that enhances the production of PGE ₂ in the living body. When this IL-1β is added to a medium containing cultured cells such as human dermal fibroblasts used in the Examples, the PGE ₂ production of the cells is enhanced and the PGE ₂ concentration in the medium is increased. However, when the prostaglandin production inhibitor of the present invention is added to the medium together with IL-1β, PGE ₂ production is inhibited. Therefore, the prostaglandin production inhibitor of the present invention suppresses an inflammatory reaction caused by increased production of PGE ₂ induced by IL-1β, and an anti-inflammatory for preventing and treating inflammatory reactions occurring at various sites. It can be used as an agent.

  The anti-inflammatory agent of the present invention containing the prostaglandin production inhibitor as an active ingredient is prepared by conventional methods such as tablets, powders, capsules, granules, fine granules, injections, inhalants, etc., or ointments, It can be made into preparations such as a skin external preparation such as a coating solution, and can be clinically used as an anti-inflammatory agent by oral or parenteral administration. The dose depends on the symptoms to be treated and the method of administration, but usually 1 μg to 10 g per day for an adult can be administered as a single dose or divided into several times a day.

As described above, the preparation forms for oral administration include tablets, powders, capsules, granules, fine granules and the like, and these preparations include pharmaceutically acceptable excipients such as starch, lactose, mannitol, Ethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose and the like are blended, and magnesium stearate or calcium stearate is added as a lubricant. Gelatin, gum arabic, cellulose ester, polyvinyl pyrrolidone and the like can be used as a binder. Examples of parenteral preparations such as injections, inhalants, and external preparations include sterile aqueous or non-aqueous solvents or emulsions. As the base of the non-aqueous solution or suspension, propylene glycol, polyethylene glycol, glycerin, olive oil, corn oil, ethyl oleate and the like are used. On the other hand, cocoa butter, macrogol and the like can be used as a base for suppositories.
Hereinafter, the effects of the present invention will be demonstrated by examples.

[Example 1]
Non-Patent Document 4 (Shoji Shibata et al., “Inhibitory Effects of Licochalcone A Isolated from Glycyrrhiza in flata Root on Inflammatory Ear Edema and Tumor Promotion in Mice”, Planta med. 57 (1991), pp 221-224) Licochalcone A was obtained according to the extraction and purification method. Using this lycochalcone A, its PGE ₂ production inhibitory activity was examined.
Normal human skin fibroblasts (quaternary culture, manufactured by Kurabo Industries) are seeded at a cell density of 1 × 10 ⁴ cells / 250 μL in a 48-well multiplate using α-minimum essential medium (α-MEM) supplemented with 10% serum. And cultured at 37 ° C., 5% CO ₂ , 95% air until confluent. After confluence, low serum culture was performed for 48 hours using α-MEM supplemented with 0.5% serum.
Thereafter, the medium was changed to 0.5% serum-added α-MEM containing 1 ng / mL of IL-1β (stimulating substance) and lycochalcone A at the concentrations shown in Table 1 below, and cultured for 24 hours as a drug treatment did. The PGE ₂ and the production amount in the culture supernatant collected after the culture were quantified by ELISA (manufactured by Cayman Chemical Co., Ltd., measured according to the manufacturer's instructions using the trade name Prostaglandin E ₂ Express EIA Kit).
In addition, the following formula, which is a well-known substance as a PGE ₂ production inhibitor

As a reference example, the same assay as in the Example was performed using a compound represented by formula (hereinafter referred to as NS-398, a commercially available product manufactured by Cayman Chemical).

  Each result is shown as an average value ± standard error as an inhibition rate (%) with respect to the control (IL-1β1 ng / mL treatment group), and a significance test was performed by Tukey's test (Tukey's multiple test). The results are summarized in Table 1 below.

From the results shown in Table 1, lycochalcone A according to the present invention inhibited the PGE ₂ production of cells induced by IL-1β stimulation in the same manner as NS-398. It is suggested that it has an IL-1β-inducible PGE ₂ production inhibitory activity.
Lycochalcone A was also demonstrated to have a strong PGE ₂ production inhibitory activity with an IC ₅₀ of 15 nM.

[Example 2]
-Inhibitory effect on PGE ₂ production by COX (cyclooxygenase) -1.
Normal human skin fibroblasts (manufactured by Kurabo Industries, Inc.) were seeded in a 48-well multiwell plate at a concentration of 1 × 10 ⁴ cells / 250 μL using α-MEM supplemented with 10% serum. The cells that reached confluence were treated with 100 nM dexamethasone for 24 hours, and after washing the cells, the test drug was incubated for 30 minutes. Thereafter, arachidonic acid (final concentration 100 μM) was added and incubated for 30 minutes, and the culture supernatant was collected. The test drugs are lycochalcone A, the COX-1 / -2 inhibitor indomethacin (Sigma) and NS-398 (NS, Cayman Chemical) known as a COX-2 selective inhibitor. The PGE ₂ content in the culture supernatant was quantified by ELISA kit (Cayman Chemical, trade name Prostaglandin E ₂ Express EIA Kit). The results are expressed as an average value ± standard error (SE) as an inhibition rate with respect to the untreated group, and a significance test was performed by Tukey's multiple test. The results are shown in Table 2.

From the results in Table 2, lycochalcone A could not suppress PGE ₂ production by COX-1 by arachidonic acid stimulation even at a concentration that could sufficiently inhibit PGE ₂ production by COX- ₂ . Similarly, NS-398, which is a COX-2 selective inhibitor, did not show a suppressive effect. On the other hand, the COX-1 / -2 inhibitor indomethacin significantly suppressed PGE ₂ production. These results suggest that lycochalcone A selectively inhibits human COX-2 derived PGE ₂ production.

[Example 3]
-Effects on COX-1,2 protein production.
Normal human skin fibroblasts seeded in a 60 mm dish and reaching confluence were treated with dexamethasone (DEX, 100 nM), NS-398 (NS, 100 nM), and lycochalcone A (Lico A, 1 μM) for 6 hours, and then the medium was removed. The cells were collected using a cell scraper with phosphate buffer. After centrifugation of the cell suspension, the collected cells were pulverized with a homogenizer, and the centrifuged supernatant was used as a COX-1 protein solution. On the other hand, the COX-2 protein solution was treated with dexamethasone (100 nM), NS-398 (100 nM) and lycochalcone A (1 μM) together with IL-1β (1 ng / mL) for 6 hours in the same manner as described above. I got it.
1 μL (protein content 50 ng) of COX-1, COX-2 standard (manufactured by Cayman Chemical) and 10 μL (protein content 30 μg) of COX-1, COX-2 protein solution were added to a 5-20% gradient SDS polyacrylamide gel (ATTO). And electrophoresis was performed with a compact PAGE (manufactured by ATTO). After electrophoresis, the gel was blotted on a PVDF membrane (ATTO) with a compact blot (ATTO). The membrane blocked with 1% skim milk (manufactured by Wako Pure Chemical Industries, Ltd.) was reacted with COX-1 polyclonal antibody (manufactured by Oxford) or COX-2 polyclonal antibody (manufactured by Cayman Chemical) at 4 ° C. overnight. After washing with 0.1% Tween-20 (Bio-Rad) -added phosphate buffer, HRP (horseradish peroxidase) labeled secondary antibody (Amersham Biosciences) was reacted at room temperature for 1 hour. After washing the secondary antibody, chemiluminescence generated for COX-1 and COX-2 proteins was detected with ECL-plus (Amersham Biosciences) by light capture (ATTO). The results are shown in FIG.

From the results of FIG. 1, the expression of COX-1 protein was not induced by IL-1β as known. In addition, the expression of COX-1 protein had no effect on the test drug containing lycochalcone A. COX-2 protein was significantly induced by IL-1β. Dexamethasone completely suppressed COX-2 protein expression. On the other hand, lycochalcone A and NS-398 had no effect on COX-2 protein expression. From these facts, it is considered that the inhibitory effect of lycochalcone A on PGE ₂ production is COX-2 selective activity inhibition similar to NS-398, unlike the steroid action that suppresses the expression of COX-2.

[Example 4]
· SPLA ₂ (secretory phospholipase _{A 2)} inhibitory activity.
The enzyme activity was measured using sPLA ₂ Assay kit (Cayman Chemical). 96-well plate placed in bee venom and test drug containing sPLA _2, the di-hepta Neu thio -PC was added as substrate and incubated for 1 hour at 37 ° C.. The color produced by the Ellman reagent (Ellman's reagent) was measured at 405nm absorbance during incubation was calculated sPLA ₂ activity from the slope. Test drug is licochalcone A, NS-398 (NS, Cayman Chemical Co.) and sPLA ₂ is active inhibitor prostaglandin Bx (PGBx, Cayman Chemical Co.). The results were expressed as an average value ± standard error (SE) as an inhibition rate with respect to the untreated group, and a significance test was performed by Tukey's multiple test. The results are shown in FIG.

From the results shown in FIG. 2, prostaglandin Bx is sPLA ₂ activity inhibitors, inhibited dose-dependently sPLA ₂ activity. Licochalcone A also showed a dose-dependent effect was significantly inhibited sPLA ₂ activity at high concentrations (100 [mu] M). On the other hand, NS-398 did not have an inhibitory effect. This result is in licochalcone A is a high concentration was found to have the effect of suppressing the sPLA ₂ activity involved in inflammatory reactions.

It is a graph which shows the result of Example 3 which concerns on this invention, and shows the expression state of COX-1 and 2 protein by each test drug (dexamethasone, NS-398, lycochalcone A). Represents the result of Example 4 according to the present invention, is a graph showing the test drug (licochalcone A, PGBx, NS-398) the inhibition of sPLA ₂ activity by.

Claims (3)

  A prostaglandin production inhibitor selected from chalcone compounds.   A prostaglandin production inhibitor comprising lycochalcone A or lycochalcone B. An anti-inflammatory agent comprising the prostaglandin production inhibitor according to claim 1 or 2 as an active ingredient.

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