WO2006123550A1

WO2006123550A1 - Prophylactic or therapeutic agent for inflammatory disease

Info

Publication number: WO2006123550A1
Application number: PCT/JP2006/309298
Authority: WO
Inventors: Nobushige Doisaki; Kiyomi Furihata; Kazuhiko Hata; Jiro Takeo; Hiroko Miyahara; Shinya Yamashita
Original assignee: Nippon Suisan Kaisha, Ltd.
Priority date: 2005-05-16
Filing date: 2006-05-09
Publication date: 2006-11-23
Also published as: JPWO2006123550A1; JP5046926B2

Abstract

[PROBLEMS] To provide a therapeutic agent for an inflammatory disease which has an anti-inflammatory effect, an anti-allergic effect and an immunosuppressive effect. [MEANS FOR SOLVING PROBLEMS] A prophylactic or therapeutic agent for an inflammatory disease which comprises 6,9,12,15-hexadecatetraenoic acid or a pharmaceutically acceptable salt or ester thereof as the active ingredient. The pharmaceutically acceptable salt of 6,9,12,15-hexadecatetraenoic acid is, for example, a sodium, potassium, magnesium, calcium, ammonium, pyridine or triethylamine salt of 6,9,12,15-hexadecatetraenoic acid. The pharmaceutically acceptable ester of 6,9,12,15-hexadecatetraenoic acid is, for example, a triglyceride, diglyceride or monoglyceride having 6,9,12,15-hexadecatetraenoic acid as the constituent fatty acid thereof or a lower alcohol ester of 6,9,12,15-hexadecatetraenoic acid.

Description

Inflammatory disease preventive or therapeutic agent

Technical field

[0001] The present invention relates to a preventive or therapeutic agent for inflammatory diseases comprising 6,9,12,15-hexadecatetetraenoic acid or a pharmaceutically acceptable salt or ester thereof as an active ingredient. , Their use for the manufacture of a medicament for the prevention or treatment of inflammatory diseases, or a method for the prevention or treatment of inflammatory diseases in which a pharmacologically effective amount thereof is administered to a human or non-human warm-blooded animal About.

Background art

[0002] Inflammation is (1) a phenomenon resulting from the body's defense reaction against pathogenic microorganisms (2) classically accompanied by redness, pain, swelling, and fever (3) loss of tissue function (4) immune system Site force-in secreted from cells mediates various reactions. The collective term for diseases associated with these conditions is inflammatory disease. Among the factors involved, cycloxygenase plays a central role in various cytokines. For inflammatory diseases, cycloxygenase inhibitors and steroids are often effective. However, in the case of chronic inflammatory diseases, there are currently limited drugs that can be used for problems such as side effects caused by long-term administration. In particular, chronic inflammatory diseases such as rheumatism, Crohn's disease, ulcerative colitis, asthma, atopic dermatitis, psoriasis, systemic lupus erythematosus and gout are recognized as typical chronic inflammatory diseases.

Crohn's disease and ulcerative colitis are diseases characterized by chronic inflammation of various parts of the digestive tract. For both diseases, sulfasalazine drugs are often effective, but no cure is known. In recent years, due to the progress of therapeutic methods, interleukin-1 blockers, antibodies against interleukin-12, and the most effective monoclonal antibody against tumor necrosis factor (THF-alpha) have been clinically applied as powerful immunoregulatory therapies. However, in these diseases, specific site force-in (TNF-alpha, IL-1, etc.) is more common than general immunosuppressive agents such as cyclosporine nyazathioprine, which may have side effects. There is a growing expectation for a therapy that suppresses the disease. Similarly, in the treatment of rheumatism, TNF-alpha antibody drugs such as infliximab have been approved, and the significance of suppressing such cytodynamic ins has been recognized.

[0003] Polyunsaturated fatty acids are the names for unsaturated fatty acids that generally have more than one double bond. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are abundant in fish oil. This corresponds to this. Polyunsaturated fatty acids are broadly classified into n-3 series such as linolenic acid, EPA, DHA, and η-6 series such as linolenolic acid, γ-linolenic acid, arachidonic acid. The position of the double bond is classified from the viewpoint of the one present at the third position from the end of the cation and the one present at the sixth position. η-6 series fatty acids are abundant in plants, while η-3 series fatty acids are abundant in aquatic organisms. Recently, η-3 and η-6 fatty acids are well balanced. It is said that it is important to take it. Fifth revised Japanese nutritional requirements In 1994, it was reported that the balance of η-6: η-3 = 4: 1 was good. Some scholars need to raise this balance to 1: 1.

[0004] Polyunsaturated fatty acids, particularly fatty acids belonging to the η-3 and η-6 strains, have various physiological functions, and research has been actively conducted in various fields. It is widely recognized that the intake of η-3 or η-6 fatty acids contained in meals is important for inflammation, immunity or allergies that are the main focus of the present invention. As arachidonic acid plays a leading role in the so-called arachidonic acid cascade and is involved in the worsening of symptoms such as inflammation and allergies as a raw material of arachidonic acid metabolites, there is a problem with overdose. η-3 fatty acids α-linolenic acid, ΕΡΑ and DHA weaken the action of arachidonic acid (specifically, it inhibits the release of arachidonic acid by cPLA2 from the cell membrane. Receptors affected by arachidonic acid metabolites) In contrast, it has been widely known that it has an action of suppressing inflammation by an action mechanism. In addition, η-3 fatty acids such as hexadecatetraenoic acid, octadecatetraenoic acid, and octadecatrienoic acid have been reported to have anti-inflammatory and antiallergic effects. Thus, many of the η-3 fatty acids have anti-inflammatory and anti-allergic effects. It is known that the η-6 fatty acids have distinctly different effects.

On the other hand, polyunsaturated fatty acids having a double bond in the carbon at the methyl terminal, ie, η-1 The family of fatty acids is a very rare fatty acid. 6,9,12,15-Hexadecatetetraenoic acid (also referred to as C1 6: 4 (n-1)) is a kind of highly unsaturated fatty acid characteristically contained in sardines and the like. Non-Patent Document 1 reports on the isolation and analysis method. 6,9,12,15- Not limited to hexadecatetraenoic acid, there are few reports on the study of bioactivity related to n-1 fatty acids.

[0006] 4,7, 10, 13-Hexadecatetetraenoic acid, an isomer of 6,9,12,15-hexadecatetetraenoic acid, is known to have various actions. In the report on the action of the n-3 fatty acid mixture, there is a report as one component in the n-3 fatty acid mixture (Non-patent Document 2 etc.) As reported as a purified single substance, 4,7,10,13-hexadecatetraenoic acid derived from seaweed is leukotrien B, leukotrien C, 5— in mouse mast cell (MCZ9). Hydroxyeiko

4 4

A report that suppresses production of satetranoic acid (Non-patent Document 3) and a report that 4,7,10,13-hexadecatetetraenoic acid derived from seaweed has cell activity (Patent Document 1) There is.

[0007] Patent Document 1: JP-A-2005-23028

Non-Patent Document 1: Journal of the American chemists' Society, 66 (9), 1323-1325, 1989. Non-Patent Document 2: Biochimica et Biophysica Acta, 1095, 187-195, 1991.

Non-Patent Document 3: Biosci. Biotechnol. Biochem., 62 (7), 1412-1415, 1998.

Disclosure of the invention

Problems to be solved by the invention

[0008] An object of the present invention is to provide a therapeutic agent for inflammatory diseases having anti-inflammatory action, anti-allergic action, and immunosuppressive action.

Means for solving the problem

[0009] As a result of intensive studies on the pharmacological action of 6,9,12,15-hexadecatetetraenoic acid, the present inventors have shown that the compound-powered interleukin, TNF-α and the like production inhibitory action The present invention was completed.

The gist of the present invention is a prophylactic or therapeutic agent for inflammatory diseases comprising 6,9,12,15-hexadecatetetraenoic acid or a pharmaceutically acceptable salt or ester thereof as an active ingredient. The gist of the present invention is a kit for the prophylaxis or treatment of inflammatory diseases comprising the above-mentioned therapeutic agent for inflammatory diseases and instructions for use.

The gist of the present invention is the use of 6,9,12,15-hexadecatetetranoic acid or a pharmaceutically acceptable salt or ester thereof for the manufacture of an agent for preventing or treating inflammatory diseases.

The present invention provides an effective amount of 6,9,12,15-hexadecatetetraenoic acid or a pharmaceutically acceptable salt or ester thereof for humans or non-humans in need of prevention or treatment of inflammatory diseases. The gist is a method for preventing or treating an inflammatory disease including administration to a warm-blooded animal. The gist of the present invention is a functional food for the prevention or treatment of inflammatory diseases comprising 6,9,12,15-hexadecatetetraenoic acid or a salt or ester acceptable as a food additive thereof as an active ingredient. To do.

The gist of the present invention is a functional food for the prevention or treatment of inflammatory diseases including the functional food for treating inflammatory diseases and instructions for use for inflammatory diseases.

Pharmaceutically acceptable salt of 6,9,12,15-hexadecatetetranoic acid in the above-mentioned inflammatory disease preventive or therapeutic agent, use, prevention or treatment method, functional food for prevention or treatment As the salt, sodium salt, potassium salt, magnesium salt, calcium salt, ammonium salt, pyridine salt, and triethylamine salt are preferable, and sodium salt is particularly preferable. The pharmaceutically acceptable ester of 6,9,12,15-hexadecatetraenoic acid is a triglyceride containing 6,9,12,15-hexadecatetraenoic acid as a constituent fatty acid. Diglycerides, monoglycerides, or lower alcohol esters of 6,9,12,15-hexadecatetetranoic acid are preferred. The present invention is effective for chronic inflammatory diseases among inflammatory diseases. The invention's effect

[0010] The 6,9,12,15-hexadecatetetraenoic acid of the present invention, or a pharmaceutically acceptable salt or ester thereof, inhibits the production of various cytodynamic ins and is anti-inflammatory. Agent Z Antiallergic agent Z It has an effective preventive or therapeutic effect on various inflammatory diseases involving inflammatory cells such as immunosuppressants.

BEST MODE FOR CARRYING OUT THE INVENTION

[0011] The 6,9,12,15-hexadecatetraenoic acid used in the present invention is a straight-chain fat having 16 carbon atoms. It is an acid and has double bonds at the 6th, 9th, 12th and 15th positions. Sometimes written as C16: 4.

The pharmaceutically acceptable salt or ester of 6,9,12,15-hexadecatetetraenoic acid of the present invention can be produced by the following method. Generally, fish oil used as a raw material for 6,9,12,15-hexadecatetraenoic acid is mostly triglyceride (TG), and 6,9,12,15-hexadecatetraenoic acid is its acyl. It exists as a residue. Fish oil also contains TGs with high melting point fatty acids such as palmitic acid and stearic acid. By cooling and crystallizing and separating these high melting point components, 6,9,12, 15-hexade force Tetraenoic acid can be concentrated. This method is called low temperature fractionation and wintering. In the case of fish oil, a method of dissolving oil in a solvent such as acetone, cooling, precipitating crystals, and separating is generally used. However, since 3 molecules of fatty acid are bound to TG, it is difficult to concentrate 6,9,12,15-hexadecatetraenoic acid to a certain level or more with TG. When highly concentrated, concentrate TG in the form of free fatty acids or monoesters. Separation of specific fatty acids from free fatty acids or monoesters can be accomplished by methods such as distillation, urea addition, column chromatography, enzymatic reaction, supercritical fluid extraction, silver complex formation, low temperature fractionation, and solvent fractionation. Fish oil contains a very wide variety of constituent fatty acids and is used in combination with multiple methods that are difficult to refine with a single purification method.

In the present invention, the pharmaceutically acceptable salt of 6,9,12,15-hexadecatetraenoic acid is preferably a pharmaceutically or physiologically acceptable alkali addition salt. For example, salts with sodium, potassium, magnesium, calcium, ammonium, pyridine, triethylamine and the like are used.

In the present invention, the pharmaceutically acceptable ester of 6,9,12,15-hexadecatetraenoic acid is 6,9,12,15-hexadecatetraenoic acid as a constituent fatty acid. The tridalylide, diglyceride, or monoglyceride contained, or the lower alcohol ester of 6,9,12,15-hexadecatetraenoic acid is preferred. As long as it contains 6, 9, 12, 15-hexadecatetraenoic acid as a constituent fatty acid of triglyceride and diglyceride, it may contain other fatty acid as a constituent fatty acid. For example, when using fish oil-derived triglycerides as raw materials, Various fatty acids with the number 14 to 22 and the number of double bonds 0 to 6 are included as constituent fatty acids, but the 6,9,12,15-hexadecatetetraenoic acid is concentrated to a concentration that allows the intake of the necessary amount. Goodbye. As the lower alcohol ester, methanol and ethanol ester are preferred.

[0013] The 6,9,12,15-hexadecatetetraenoic acid, or a pharmaceutically acceptable salt or ester thereof used in the present invention is administered in various forms. The dosage form is determined according to various preparation forms, no particular limitation, age, sex and other conditions of the patient, degree of disease, etc. For example, tablets, pills, powders, granules, syrups, solutions, suspensions, emulsions, granules and capsules are orally administered. In the case of injections, they are administered alone or mixed with normal replacement fluids such as glucose and amino acids and administered intravenously, and if necessary, administered alone intramuscularly, intradermally, subcutaneously, or intraperitoneally. Is done. In the case of suppositories, it is administered rectally. Oral administration is preferred. 6,9,12,15-Hexacatecatelaenoic acid is easy to oxidize! / Since it is a liquid oil, it needs to be treated to prevent acidification, such as by filling capsules and antioxidants.

[0014] These various preparations are prepared in accordance with conventional methods. The main ingredients are excipients, binders, disintegrants, lubricants, solubilizers, corrigents, flavoring agents, coating agents, sweeteners, colorants, lubricants. , Stabilizers, preservatives

In addition, it can be formulated using known adjuvants that can be usually used in the field of pharmaceutical preparations such as preservatives. In molding into a tablet form, a carrier conventionally known in this field can be widely used. For example, lactose, sucrose, sodium chloride sodium salt, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, key Excipients such as acids, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polypyrrole pyrrolidone sugar binder, dried starch, Sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose and other disintegrants, sucrose, stearin, cocoa butter, hydrogen Disintegration inhibitors such as additive oils, Absorption accelerators such as ammonium base and sodium lauryl sulfate, humectants such as glycerin and starch, adsorbents such as starch, lactose, kaolin, bentonite and colloidal key acid, purified talc, stearate, boric acid powder, polyethylene glycol Examples thereof include lubricants such as Furthermore, tablets are tablets with a normal skin if necessary. For example, sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multilayer tablets can be used.

[0015] When forming into the form of a pill, those conventionally known in this field can be widely used as a carrier. For example, excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, and talc Examples thereof include binders such as gum arabic powder, tragacanth powder, gelatin and ethanol, and disintegrants such as laminaran strength.

In molding into a suppository, conventionally known carriers can be widely used as carriers, such as polyethylene glycol, cocoa butter, higher alcohols, higher alcohol esters, gelatin, semi-synthetic glycerides and the like. Can be mentioned.

When prepared as injections, solutions and suspensions should be sterilized and should be isotonic with blood. Any of those commonly used in this field can be used, such as water, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxyisostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. Can do. In this case, a sufficient amount of sodium chloride, glucose, or glycerin to prepare an isotonic solution may be contained in the pharmaceutical preparation. Ordinary solubilizers, buffers, soothing agents, etc. May be added. Furthermore, if necessary, colorants, preservatives, fragrances, flavoring agents, sweetening agents, and other pharmaceuticals may be included.

[0016] The amount of the active ingredient-compound contained in the above pharmaceutical preparation is not particularly limited and is appropriately selected over a wide range. When the capsule is filled, liquid 6,9,12,15-hexa It is possible to fill only with decatetetranoic acid, and when other preparations are used, it is appropriate that the amount is usually 1 to 70% by weight in the total composition. In the present invention, the dose of 6,9,12,15-hexadecatetetraenoic acid or a pharmaceutically acceptable salt or ester thereof varies depending on the symptom, age, administration method, etc. In some cases, it is desirable to administer 1 to 1200 mg / l, preferably 50 to: LOOOmg in one or several divided doses depending on the symptoms. In the case of intravenous administration, it is desirable to administer 0.01 to 500 mg, preferably 0.1 to 200 mg per day to adults in one or several divided doses according to symptoms. [0017] Functional foods containing 6,9,12,15-hexadecatetraenoic acid may be in the form of capsules, tablets, beverages such as milk, soy milk, soft drinks, or foods It is also possible to have an accompaniment.

[0018] The preventive or therapeutic agent for inflammatory diseases of the present invention has an anti-inflammatory action, an anti-allergic action, and an immunosuppressive action by mechanisms such as TNF-a production inhibition and cytoforce-in production inhibition action. Effective for various inflammatory diseases. Particularly suitable for chronic inflammatory diseases. Examples include ulcerative colitis, inflammatory bowel diseases such as Crohn's disease, arthritis such as rheumatism, and the like.

6,9,12,15-Hexadecatetetraenoic acid is a fatty acid contained in fishes such as sardines, dicins and menhadens that have been ingested by mankind for many years as a dietary habit (Reference; RGA ckman: Fatty Acid Composition of Fish Oils, In Nutritional Evaluation of Long—Chai n Fatty Acid in Fish Oil (Edited by SM Barlow & ME Stansby: Academic Press, London, New York, (1982) pp. 25-88). RG Ackman, CA Eaton & J. Hingle y: Fillet Fat and Fatty Acid Details for Newfoundland Winter Herring, Canadian Ins titute of Food Science and Technology Journal, 8 (3), 155-159 (1975)), high safety it is conceivable that.

Examples of the present invention will be described below, but the present invention is not limited to these.

Example 1

[0020] Evaluation of 6,9,12,15-hexadecatetraenoic acid activity using an in vitro cultured cell line

The inhibitory effect of 6,9,12,15-hexadecatetetraenoic acid (C16: 4) on the production of various cyto force-ins was evaluated using the Atsy system of cultured cells. In the TNF-α test, a comparative test was conducted using eicosapentaenoic acid (ΕΡΑ), which is a fatty acid of 20 carbon atoms, 4 double bonds, and η-3.

B, J.

Atsay method

Human peripheral blood mononuclear cells ( ⁵ X loVml) and drug are pre-cultured in AIM-V medium (pH 7.4) for 30 minutes. Stimulate cells by adding stimulant (concanavalin-8; 20 or lipopolysaccharide; 25 gZml) and incubate overnight at 37 ° C, 5% CO. Various site forces in the culture supernatant In levels were measured by sandwich ELISA. The drug concentration was screened at 30, 10, 3, 1, 0.3 μΜ, and the inhibition of production of various site force ins was evaluated by IC.

50

The 6,9,12,15-hexadecatetraenoic acid used in this example is an ethyl ester (purity 96%) produced by the method of Example 3.

The results are shown in Table 1. 6,9,12,15-Hexadecatetraenoic acid (C16: 4) showed an inhibitory effect on any site strength. Eicosapentaenoic acid was effective in producing TNF-α even at a concentration of 30 mg.

[0021] [Table 1]

From the above results, it was shown that 6,9,12,15-hexadecatetraenoic acid has an effect of suppressing the production of various cytostatic ins. These results suggest that 6,9,12,15-hexadecatetetraenoic acid has anti-inflammatory, anti-allergic and immunosuppressive effects. Among the above cytokines, interleukin-2 and TNF-α are factors that are particularly important for their association with various diseases.

Example 2

[0022] Activity report of 6.9.12.15-hexadecatetrasuccinic acid used in inflammation model

1. Inflammatory colitis model-1

A group of 5 male Wistar rats weighing 200 ± 10g fasted for 24 hours. Peripheral colitis was induced by enteral administration of DNBS (2, 4-Dinitrobenzensulfonic acid, 30 mg in 0.5 ml 30% ethanol). The compound (dissolved in 2% Polygly ester solution) was orally administered once daily for 7 days during the study period. DNBS was enterally administered 24 hours after the first drug administration and 2 hours after the second drug administration to induce colitis. Control group has similar time DNBS was administered. The other control group is a group not administered with DNBS. After continuous administration of the drug for 7 days, the large intestine was isolated from rats that were appropriately lethal at 24 hours, and the weight was measured. Diarrhea status was monitored throughout the study. When the abdominal cavity was opened, the adhesion between the large intestine and other organs was observed, and further ulcers of the large intestine were evaluated. The ratio of the large intestine to body weight was calculated for each animal and calculated by the following formula.

(A) Large intestine weight per 100 g of body weight = isolated large intestine X 100 / day 8 body weight

(B) Large intestine increase per 100g body weight = (A) Large intestine increase per 100g body weight of untreated control

(C) Large intestine weight decrease rate per 100g body weight = large increase in large intestine per 100g body weight of DNBS control (B) I large increase in large intestine per 100g body weight in DNBS control In this study, 6, 9, 12, 15- As the oxadecatetraenoic acid, the ethyl ester (purity 78%) produced by the method of Example 3 was used.

The results are shown in Table 2. The weight of the large intestine per 100g body weight of the untreated control group is 0.

Compared to 293 g, administration of DNBS increased the colon weight to 0.784 g. This indicates that DNBS treatment caused inflammation in the large intestine. Administration of 6,9,12,15-hexadedecatetraenoic acid (C16: 4 n-1) reduced the increase in colon weight to 0.656 g by oral administration of 100 mg / kg. The rate of decrease was 26%. In the case of oral administration of 300 mg / kg, the increase in the weight of the large intestine was reduced to 0.6 g. The decrease rate was 37%. As a positive control, the effect of Sulfasalazine was examined. Sulfasalazine orally administered 300 mg / kg reduced the increase in colon weight to 0.582 g. The decrease rate was 41%. As a result of t-test, there was a significant difference of P <0.05 between the 100 mg and 300 mg groups of 6,9,12,15-hexadecatetetraenoic acid and the control group administered with DNBS alone. Was recognized. In addition, the Sulfasalazine 300 mg administration group showed a significant difference at P 0.01 compared to the control group to which DNBS alone was administered.

[0024] [Table 2] (A) (0

Dose per 100g body weight Large drug per 100g body weight

(mg / kg) colon weight weight 誦 difference intestinal increase weight decrease rate

N (g) (%)

Untreated control 5 0. 293 0. 008

DNBS control 5 0. 784 0. 026 0

016: 4 n-1 5 100 0. 656 0. 034 26 *

016: 4 n-1 5 300 0. 6 0. 047 37 *

Sulfasalazine 5 300 0. 582 0. 029 41

P <0. 05, Ρ <0. 01

[0025] Thus, 6,9,12,15-hexadecatetetraenoic acid has almost the same effect as Sulfasalazine, a pharmaceutical agent that is currently used in clinical practice, and has similar administration. In quantity in a rat inflammatory colitis model. This result indicates that it can be a therapeutic agent for Crohn's disease and ulcerative colitis, which are human diseases similar to 6,9,12,15-hexadecatetetraenoic acid.

[0026] 2. Inflammatory colitis model-II

In a similar model, the effects of 6,9,12,15-hexadecatetraenoic acid and EPA were compared.In this study, 6,9,12,15-hexadecatetraenoic acid was Ethyl ester (purity 96%) produced by the method was used, and EPA used triglyceride (lipid composition: triglyceride 99%, fatty acid composition: EPA 98%).

The results are shown in Table 3. The weight of the large intestine per 100 g of body weight in the untreated control group was 0.225 g, whereas when DNBS was administered, the large intestine weight increased to 0.704 g. This indicates that DNBS treatment caused inflammation in the large intestine. Administration of 6,9,12,15-hexadecatetetranoic acid reduced the increase in colon weight to 0.574 g at 100 mg / kg orally. The decrease rate was 27%. This shows that the test results in the previous section were almost certainly reproduced. On the other hand, when EPA was administered orally at 100 mg / kg, the increase in the weight of the large intestine was reduced to 0.607 g. The rate of decrease was 20%. As a positive control, the effect of Sulfasalazine was examined. Sulfasalazine decreased the increase in colon weight to 0.574 g by oral administration of 300 mg / kg I let you. The reduction rate was 35%. As a result of the t-test, there was a significant difference of P 0.01 between the 100 mg-treated group of 6,9,12,15-hexadecatetetraenoic acid and the control group administered with DNBS alone. . In addition, the Sulfasalazine 300 mg group showed a significant difference at P 0.01 compared to the control group administered with DNBS alone. There was no significant difference between the EPA-treated group and the control group treated with DNBS at a 5% risk.

These results indicate that not all fatty acids can be effective in such anti-inflammatory models, especially polyunsaturated fatty acids, especially 6,9,12,15-hexadeca. This suggests that tetranoic acid has a strong anti-inflammatory effect. In addition, EPA is the first highly unsaturated fatty acid that has been widely studied to exhibit anti-inflammatory effects. This test can be considered as the first test result that has found the possibility of having a stronger anti-inflammatory effect than 6,9,12,15-hexadecatetraenoic acid EPA.

[Table 3]

P <0. 01

3. Carrageenan edema model

A group of 6 male ICR mice weighing 22 ± 2g fasted for 24 hours. Carrageenan (50 μl of 1% suspension) is injected into the paw 1 hour after oral administration of the drug. The hind limb edema was used as an index of inflammation, and was measured with a dedicated measuring instrument 4 hours after carrageenan administration. This study is a very acute model that analyzes the effects of a single dose of 6,9,12,15-hexadecatetetraenoic acid on edema formation. In this test, 6,9,12,15-hexadecatetraenoic acid was used as the ethyl ester (purity 78%) prepared by the method of Example 3. The results are shown in Table 4. In the control group, hind paw volume increased to 5.7 (X 0.01 ml) due to edema caused by carrageenan administration. In the group in which 6,9,12,15-hexadecatetraenoic acid was administered 1 hour before carrageenan administration, the volume of the hind paws was suppressed to 4.5 (X 0.01 ml). With the positive control aspirin, the hind paw volume was pushed to 3.3 (X 0.01 ml). Thus, in an animal model that is an acute inflammation model, 6,9,12,15-hexadecatetetranoic acid has been shown to suppress inflammation even after a single administration.

[Table 4]

P

4. Monoclonal antibody-induced collagen arthritis

A group of 5 BALB / cByJ mice weighing 20 ± 2 g. Four types of monoclonal antibodies against type II collagen (D8, F10, DI-2G, A2) were administered intravenously at 20 mg per mouse on day 0. On the third day, lipopolysaccharide (LPS, 25 μg / mouse) was intravenously administered to induce inflammation. The drug was orally administered once a day for 3 days from 1 hour after LPS administration on the third day. On the third day after LPS administration, hind paw edema was measured with a dedicated measuring instrument. In this test, 6,9,12,15-hexadecatetraenoic acid was an ethyl ester produced by the method of Example 3 (purity 78%).

The results are shown in Table 5. In the control group, the volume of the hind paws increased to 10 (X 0.01 ml) by administration of four monoclonal antibodies against type II collagen and edema caused by induction of LPS. In the group that received oral administration of 6,9,12,15-hexadecatetraenoic acid 1 hour after LPS once a day for 3 days, the hind paw volume was kept at 5.6 (X 0.01 ml). It was. The reduction rate was 44%, and a significant difference was observed by t-test (P 0.05) .As such, in this model, 6,9,12,15-hexadecatetetraenoic acid Is shown to suppress inflammation [Table 5]

* P 0. 05

5. Analgesic effect of 6,9,12,15-hexadecatetraenoic acid on rat beer yeast inflammation foot pain (Randall-Selitto method)

6,9,12,15-Hexadecatetetraenoic acid (using the ethyl ester produced by the method of Example 3 (purity 91.5%)) (specific gravity 0.9) in a 833 mg polypropylene centrifuge tube, 3 A 25% polyglycerin ester aqueous solution was added and emulsified to prepare a test solution (30 mg / mL). The test solution was administered by the oral route. Administration was performed once a day for 7 days. The dose of 6,9,12,15-hexadecatetraenoic acid was 300 mg / kg, and the dose volume was 10 mL / kg. The control group received the same volume of vehicle as the test solution. 1 g of beer yeast (manufactured by Yeast, Brewers Bottom, Sigma) was weighed, suspended in a physiological saline solution, and made up to 10 mL to prepare a 10 w / v% beer yeast physiological saline solution. One hour after the final administration of the test solution, inflammation was induced by subcutaneously administering 0.1 mL / mouse of 10 w / v% Brewers yeast physiological saline subcutaneously on the right hind footpad of the animal. At the three time points before administration of the test solution, 1 hour before and 4 hours after the final administration of the test solution (3 hours after administration of 10 w / v% Brewers yeas t physiological saline), the right hind paw of the rat was Pressure threshold was applied by TK-201, Newcom), and the pain threshold was measured.

The representative value of each group was expressed by the average value and standard error of the pain threshold. The equivariance was tested by the F test between the control group and the test solution group. The number of animals used is 10 in each group.

The results are shown in Table 6. The average pain threshold (mean ± standard error) of the control group before the start of test solution administration, 1 hour before the last administration of the test substance and 3 hours after the inflammation (4 hours after the last administration of the test substance) is 92 ± 1 mmHg , 90 ± 1 mmHg &, 39 ± 3 mmHg, and the average nociceptive response after 3 hours of inflammation decreased by 51 ± 3 mmHg.

6,9,12,15-hexadecatetraenoic acid group before administration of test solution, final administration of test substance The mean pain threshold at 1 hour before and after 3 hours of inflammation was 92 ± 1 mmHg, 89 ± 2 mmHg, and 52 ± 3 mmHg, and the average pain response at 3 hours after inflammation decreased by 37 ± 2 mmHg. On the other hand, a significant high value (p <0.01) and a significant (p <0.01) low value were observed in the pain threshold at 3 hours after inflammation.

[0033] [Table 6]

: P <0.01

Example 3

[0034] 1. Distillation

Distillation of sardine oil ethyl ester (containing 1.9% 6,9,12,15-hexadecatetraenoic acid ethyl ester) converted to ethyl ester using sodium ethylate under conditions of 135 ° C and O.lmmHg, A fraction containing 4.25% 6,9,12,15-hexadecatetraenic acid ethyl ester was obtained.

2. Urea addition

60.5 g of the obtained fraction was added dropwise to a 60 ° C methanol solution (600 ml) in which 180.0 g of urea was dissolved. After stirring for 1 hour, the mixture was further stirred at 4 ° C for 16 hours, and the precipitated crystals were sucked. It was removed by filtration. The methanol in the obtained solution was distilled off, and then partitioned with 400 ml of hexane and 400 ml of distilled water, and the obtained hexane layer was further washed with 400 ml of distilled water three times. In addition, the aqueous layer obtained at the first time was extracted four times with 400 ml of hexane. Combine all hexane layers, dehydrate with anhydrous magnesium sulfate, filter anhydrous magnesium sulfate, distill off hexane, contain 17.1% 6,9,12,15-hexadecatetraenoic acid ethyl ester 14.0 g of urea treated oil was obtained. The recovery rate of 6,9,12,15-hexadecatetraenoic acid ethyl ester in the urea addition treatment was 92.6%. Urea addition treatment under the same conditions was further repeated 10 times to obtain a urea-treated oil containing 17.4% of 6,9,12,15-hexadecatetraethyl ester.

3.Column The obtained 6,9,12,15-hexadecatetraethyl ethyl ester 17.4% urea-treated oil 35g was filled with octadecylsilyl silica gel (Fuji Silysia Chemical Co., Ltd., 100-200mesh). Attached to an open column (7.5 cm φ X 25 cm), 800 ml was passed through 95% methanol, and 200 ml fractions were collected. As a result of distilling off the solvent of the obtained fraction, 1.3 g of 6,9,12,15-hexadecatetetraenoic acid ethyl ester (recovery rate: 17.5%) was obtained from Fraction 3. In the other fractions, 5.3 g of 6,9,12,15-hexadecatetraethyl ethyl ester (recovery rate 51.6%) was 59.5% from fraction 4, and 26.4% of 6,9,12,15-hexadeca was 52.5% from fraction 5. As a result, 6.0 g of tetraethyl ethyl ester (recovery rate 26.2%) was obtained, and the total recovery rate was 95.3%.

Column treatment under the same conditions was repeated several times, and fractions with low purity were collected to obtain 31.2% of 6,9,12,15-hexadecatetraenoic acid ethyl ester. As a result, 2.3 g (yield 21.1%) of 9,4.7% of 6,9,12,15-hexadecatetraenoic acid ethyl ester was obtained. In other fractions, 58.2% from fraction 4, 6,9,12,15-hexadecatetraenic acid ethyl ester lO.lg (recovery 56.3%), 26.3% from fraction 5, 6,9,12,15 -8.7 g of hexadecatetetraenoic acid ethyl ester (recovery rate 21.9%) was obtained, and the total recovery rate was 99.2%.

[0035] 96.0% of 6,9,12,15-hexadecatetraenoic acid ester prepared in the same manner as described above was analyzed by 400 MHz 1H-NMR and GC-MS. It was confirmed that it was 6,9,12,15-hexadecatetraenoic acid.

1H-NMR (400MHz, CDC13): 1.25 (t, J = 7.1Hz, 3H), 1.38 (m, 2H), 1.6 (m, 2H), 2.1 (m, 2H), 2.30 (t, 2H), 2.8 (m, 6H), 4.12 (quart., J = 7.1Hz, 2H), 5.0 (m, 2H), 5.3—5.4 (m, 6H), 5.7 (m, 1H)

Mass m / z: 276 (M +.), 261, 247, 235, 208, 189, 171, 161.

Example 4

[0036] ¾ Method of reporting by Takatsuki liquid chromatograph

A high-performance liquid chromatograph was used. 200 μ 1 of 6,9,12,15-hexadecatetraenoic acid ethyl ester (34.5%) in methanol (200 μ1) (approx. 90 mg as an oil) Cosmo Seal 5C18-AR packed column (20.0 mmI. D. X 250mm) Of the body chromatograph (solvent methanol, flow rate 5 ml / h), 95.4% of 6,9,1 2,15-hexadecatetraenoic acid ethyl ester 18.7 mg (recovery 57.3%) Obtained.

94.5% 50% methanol solution of 9,9,12,15-hexadecatetraenoic acid ethyl ester obtained by several fractions by high performance liquid chromatograph under the same conditions 200 1 (about 90m as oil g) was again fractionated under the same conditions, 99.1% 6,9,12,15-hexadecatetraenoic acid ethyl ester 15.9 mg (recovery 18.5%), 97.5% 6,9,12 , 15-hexadecatetraethyl ester 28.1 mg (recovery 32.2%) was obtained.

Industrial applicability

A prophylactic or therapeutic agent for inflammatory diseases or diseases caused by inflammatory diseases such as colitis and arthritis can be provided. In addition, since it is a fatty acid derived from food and high safety can be expected, a functional food having a function of preventing or treating inflammatory diseases can be provided.

Claims

The scope of the claims

[I] A prophylactic or therapeutic agent for inflammatory diseases comprising 6,9, 12, 15-hexadecatetetraenoic acid or a pharmaceutically acceptable salt or ester thereof as an active ingredient.

[2] 6,9,12,15-Hexadecatetetraenoic acid pharmaceutically acceptable salt is sodium salt, potassium salt, magnesium salt, calcium salt, ammonium salt, pyridine salt The prophylactic or therapeutic agent for inflammatory diseases according to claim 1, which is any one of the following salts: triethylamine salt.

[3] a triglyceride in which the pharmaceutically acceptable ester of 6,9,12,15-hexadecatetraenoic acid contains 6,9,12,15-hexadecatetraenoic acid as a constituent fatty acid, 2. The preventive or therapeutic agent for inflammatory diseases according to claim 1, which is diglyceride, monoglyceride, or a lower alcohol ester of 6,9,12,15-hexadecatetetranoic acid.

[4] The inflammatory disease preventive or therapeutic agent according to claim 1, 2 or 3, wherein the inflammatory disease is a chronic inflammatory disease.

[5] Claim 1! 4 A kit for the prevention or treatment of inflammatory diseases including a therapeutic agent for any of inflammatory diseases and instructions for use.

[6] Use of 6,9,12,15-hexadecatetetranoic acid or a pharmaceutically acceptable salt or ester thereof for the manufacture of an agent for preventing or treating inflammatory diseases.

[7] 6,9,12,15-Hexadecatetetraenoic acid pharmaceutically acceptable salt is sodium salt, potassium salt, magnesium salt, calcium salt, ammonium salt, pyridine salt 7. Use according to claim 6, wherein the salt is triethylamine salt.

[8] A triglyceride in which the pharmaceutically acceptable ester of 6,9, 12, 15-hexadecatetraenoic acid contains 6,9, 12,15-hexadecatetraenoic acid as a constituent fatty acid, 7. The use according to claim 6, which is a diglyceride, a monoglyceride, or a lower alcohol ester of 6,9,12,15-hexadecatetetranoic acid.

9. Use according to claim 6, 7 or 8, wherein the inflammatory disease is a chronic inflammatory disease.

[10] An effective amount of 6,9,12,15-hexadecatetetraenoic acid or a pharmaceutically acceptable salt or ester thereof or a human or a person in need of prevention or treatment of inflammatory disease A method for preventing or treating an inflammatory disease comprising administering to a non-human warm-blooded animal.

[II] 6,9,12,15-Hexadecatetetranoic acid pharmaceutically acceptable salt is sodium salt, potassium 11. The method for prevention or treatment according to claim 10, wherein the salt is any one of um salt, magnesium salt, calcium salt, ammonium salt, pyridine salt and triethylamine salt.

[12] a triglyceride in which the pharmaceutically acceptable ester of 6,9, 12, 15-hexadecatetraenoic acid contains 6,9, 12,15-hexadecatetraenoic acid as a constituent fatty acid, 11. The prevention or treatment method according to claim 10, which is a diglyceride, a monoglyceride, or a lower alcohol ester of 6,9,12,15-hexadecatetetranoic acid.

[13] The method according to claim 10, 11 or 12, wherein the inflammatory disease is a chronic inflammatory disease.

[14] A functional food for preventing or treating inflammatory diseases comprising 6,9, 12, 15-hexadecatetetraenoic acid or a salt or ester acceptable as a food additive thereof as an active ingredient.

[15] 6,9,12,15-Hexadecatetetraenoic acid pharmaceutically acceptable salt is sodium salt, potassium salt, magnesium salt, calcium salt, ammonium salt, pyridine salt 15. The functional food for preventing or treating inflammatory diseases according to claim 14, which is any one of salts of triethylamine.

[16] a triglyceride in which the pharmaceutically acceptable ester of 6,9, 12, 15-hexadecatetraenoic acid contains 6,9, 12,15-hexadecatetraenoic acid as a constituent fatty acid, 15. The functional food for preventing or treating inflammatory diseases according to claim 14, which is a diglyceride, a monoglyceride, or a lower alcohol ester of 6,9,12,15-hexadecatetetranoic acid.

[17] The functional food for preventing or treating inflammatory disease according to claim 14, 15, or 16, wherein the inflammatory disease is a chronic inflammatory disease.

[18] A functional food for the prevention or treatment of inflammatory diseases, comprising a functional food for treating any inflammatory disease, and instructions for use for inflammatory diseases.