JP3233709B2 - Immunomodulator - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a low-toxicity immunomodulator.

[0002]

The compound of the present invention, 3-hydroxymethyl-6
-Methoxy-8-hydroxy-1H-2-benzopyran-1-one is
Antibiotic MI43 with anticancer action in JP-A-3-2177
Although it is disclosed as -37F11, it is not described in the specification to have an immunomodulatory effect.

On the other hand, drugs such as steroid hormones, gold preparations, D-penicillamine, levamisole, and salazosulfapyridine used for the treatment of autoimmune diseases are not suitable for adrenal insufficiency, infectious disease, renal disorder, hematopoietic disorder, Since it sometimes causes serious side effects such as gastrointestinal disorders, it is a great limitation in use.

[0004]

DISCLOSURE OF THE INVENTION The present invention provides an immunomodulator having excellent immunomodulatory activity, low toxicity and safe use.

[0005]

Means for Solving the Problems The present inventors have been diligently studying the pharmacological activity of 3-hydroxymethyl-6-methoxy-8-hydroxy-1H-2-benzopyran-1-one. The present inventors have found that the substance has an immunomodulatory activity including an anti-rheumatic activity and completed the present invention. The present invention is an immunomodulator containing 3-hydroxymethyl-6-methoxy-8-hydroxy-1H-2-benzopyran-1-one represented by the following formula (I) as an active ingredient.

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The compound (I) of the present invention is a microorganism belonging to the genus Streptoverticillium and having the ability to produce the compound (I), for example, Streptoverticillium eurosidicum strain MI43-37F11 (FERM BP- 2783) is cultured in a nutrient medium and collected from the culture solution (JP-A-3-2177). Further, the compound (I) of the present invention is a 2-benzopyran derivative, for example, 3-benzyloxymethyl-6,8-dimethoxy-1H-2-benzopyran-1-one, which is reacted with BBr under a nitrogen stream.
₃ can be produced by reacting
No. 4-112884).

The compound (I) of the present invention has an inhibitory effect on adjuvant arthritis and collagen-induced arthritis as shown below, and exhibits rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, periarteritis nodosa and juvenile It is expected to be applicable to autoimmune diseases such as diabetes. In addition, it has almost no toxicity and can be used effectively and safely.

(A) Inhibitory effect of adjuvant arthritis The preventive effect of adjuvant arthritis on female 9-week-old female Lewis
The rats were examined using 6 to 7 rats per group. That is, adjuvant arthritis was induced by inoculating 0.1 ml of killed Mycobacterium tuberculosis (6 mg / ml) suspended in liquid paraffin into the skin of the foot pad of the right hind leg of anesthetized ether rats. Compound (I) was orally administered at 10, 30, and 100 mg / kg, respectively, once a day for 20 days from the day of sensitization with the adjuvant. The inhibitory effect of adjuvant arthritis was evaluated by using a digital caliper to periodically measure the thickness of the foot pads of the hind limb of the sensitized site and the non-sensitized site. Table 1 shows the results.

[0009]

[Table 1]

The compound (I) of the present invention, when administered orally at a dose of 10 to 100 mg / kg / day, significantly increases the swelling of the hind limb of the non-sensitized site of adjuvant arthritis as seen characteristically in many immunomodulators. Suppressed. The 100 mg / kg / day test group of compound (I) had a greater inhibitory effect than the same dose of salazosulfapyridine used clinically for the treatment of rheumatoid arthritis.

(B) Inhibition of collagen-induced arthritis The effect of preventing the onset of collagen-induced arthritis by 6-8 DBAs per group
/ 1J mice were examined. That is, type II collagen was emulsified with an equal volume of Freund's complete adjuvant to prepare a 1 mg / ml administration solution. The mice were sensitized by injecting 0.1 ml thereof into the skin at the ridge of the mouse. Twenty-one days later, 0.1 ml of type II collagen emulsified in the same manner as described above was administered intraperitoneally to mice to perform booster immunization to induce arthritis. The compound (I) was orally administered at 30 and 100 mg / kg and the control drug salazosulfapyridine at 100 mg / kg once a day for 43 days from the first sensitization day of type II collagen. The inhibitory effect of collagen-induced arthritis was evaluated by periodically measuring the thickness of the hind limb foot pad using a digital caliper. Table 2 shows the results.

[0012]

[Table 2]

The compound (I) of the present invention contains 30 and 100 mg / kg /
Oral administration on the day significantly suppressed hind limb swelling due to collagen-induced arthritis. The 100 mg / kg / day test group of compound (I) had a greater inhibitory effect than the same dose of salazosulfapyridine.

(C) Oral acute toxicity The compound of the present invention was administered to 7 ICR mice and 7 SD rats each.
(I) was orally administered at 2000 mg / kg and 1000 mg / kg, respectively, but no individual died and no toxic symptoms were observed. The toxicity of the compound (I) of the present invention to warm-blooded animals,
Very low.

The compound (I) of the present invention is useful as an immunomodulator, and its pharmaceutical preparation can be prepared by using known fillers, bulking agents,
It can be prepared using a diluent or excipient such as a binder, a humectant, a disintegrant, a disintegration inhibitor, a surfactant and a lubricant. Various forms can be selected as the pharmaceutical preparation depending on the purpose of treatment, and typical examples are tablets, pills, powders, solutions, suspension syrups, emulsions, granules, capsules, suppositories, injections Agents (solutions, suspensions, etc.).

For molding into tablets, known carriers can be widely used, such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silica, etc. Agent,
Water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, binders such as carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, dried starch, sodium alginate, agar powder, laminaran powder, hydrogen carbonate Sodium, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride stearate, starch, lactose, etc., disintegrators such as sucrose, cocoa butter, hydrogenated oil, quaternary ammonium base, lauryl Absorption promoters such as sodium sulfate; humectants such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid; lubrication such as purified talc, stearate, borate powder, and polyethylene glycol Agents are examples It can be.

Further, the tablets can be made into tablets coated with a usual coating, if necessary, such as sugar-coated tablets, gelatin-encapsulated tablets, film-coated tablets, double tablets and multilayer tablets. In the case of molding into a pill form, those conventionally known in the art can be widely used as a carrier, for example, excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, talc, gum arabic powder, Examples thereof include binders such as tragacanth powder, gelatin and ethanol, and disintegrants such as laminaran and agar.

For molding into a suppository form, carriers conventionally known in the art can be widely used, and examples thereof include polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides. be able to.

When prepared as an injection, the solution and suspension are preferably sterilized and isotonic with blood. When molded into the form of a solution or suspension, the solution or suspension is used as a diluent. Any of those commonly used in the field can be used, and examples thereof include water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters.
In this case, a sufficient amount of salt, glucose or glycerin to prepare an isotonic solution may be contained in the preparation, and a usual solubilizing agent, a buffer, a soothing agent and the like may be used. Is also good. For oral administration, a coloring agent, a preservative, a flavoring agent, a flavoring agent, a sweetening agent or the like and other pharmaceuticals may be contained in the preparation as required.

The amount of the compound (I) of the present invention to be contained in the immunomodulator of the present invention is not particularly limited and may be selected from a wide range, but is usually a solid preparation such as tablets, granules and capsules. Is 1 to 70% by weight, preferably 5 to 50% by weight of the total composition, and in the case of liquid preparations such as liquid preparations, injection preparations and suspension preparations,
0.1 to 10%.

The method of administration of the compound (I) of the present invention is not particularly limited, and the compound (I) is administered by a method according to various preparation forms, age, sex and other conditions of the patient, degree of disease and the like. For example,
In the case of tablets, pills, solutions, suspension syrups, emulsions, granules and capsules, they are orally administered. In the case of injections, they are administered intravenously, alone or as a mixture with a normal replenisher such as glucose or amino acid, and if necessary, intramuscularly, intradermally, subcutaneously or intraperitoneally. In the case of suppositories, they are administered rectally.

The dose of the compound (I) of the present invention can be appropriately increased or decreased depending on the usage, age and sex of the patient, other conditions, the degree of the disease and the like. In the case of oral administration, 0.3 to 300 mg per day In the case of parenteral administration, 0.03 to 30 mg / kg is appropriate.

Next, the present invention will be described more specifically with reference to examples.

【Example】

Example 1 Capsule An immunomodulator having the following composition and an excipient are uniformly mixed.
The obtained mixture is filled into about 200 mg per capsule into a No. 3 hard gelatin capsule.

[0024]

[Table 3]

Example 2 Tablets The immunomodulator is mixed well with lactose and half the amount of corn starch in the following mixing ratio. The mixture is granulated by mixing with 10% starch paste and sieving. The rest of the corn starch and magnesium stearate are added and mixed well to form a tablet having a diameter of 8 mm and a weight of 250 mg.

[0026]

[Table 4]

Example 3 Suspension Syrup The immunomodulator is suspended in the following mixing ratio and placed in a dosing bottle.

[0028]

[Table 5]

Reference Example 3-hydroxymethyl-6-methoxy-
Production of 8-hydroxy-1H-2-benzopyran-1-one Galactose 2.0%, Dextrin 2.0%, Soypeptone (Bacto soytone manufactured by Difco) 1.0%, Corn steep liquor (Nippon Shokuhin Kako) 0.5%, Ammonium sulfate 0.2 Liquid medium (pH 7.4) consisting of 0.003%, a defoaming silicone oil (Shin-Etsu Chemical Silicon KM70) 0.003%
Three Erlenmeyer flasks with waffles dispensed by 0 ml were inoculated with one loop of streptoverticillium eurosidicum MI43-37F11 strain (FERM BP-2783) cultured on an agar slant medium at 30 ° C. for 2 days. Shaking culture was performed to prepare a seed culture solution.

Next, glycerin 2.0%, essan meat
(Manufactured by Ajinomoto Co.) 1.5%, monopotassium phosphate 0.1%, cobalt chloride hexahydrate 0.0005%, defoaming silicone oil 0.00
Eighty Sakaguchi flasks were prepared by dispensing 125 ml of a liquid medium containing 3% and adjusted to pH 6.2 with dipotassium phosphate and inoculated with 2.5 ml of a seed culture solution, and cultured at 28 ° C. for 4 days. .

After completion of the fermentation, the culture solution was filtered, and the obtained filtrate was adjusted to pH 5 and extracted with an equal amount of ethyl acetate. It was concentrated under reduced pressure to obtain 2.0 g of a brown substance. This was adsorbed on a 70 ml silica gel column packed with n-hexane, washed with 300 ml of n-hexane-ethyl acetate (8: 2), and then eluted with n-hexane-ethyl acetate (1: 1). The eluate was concentrated under reduced pressure, and the obtained oil was again adsorbed to a silica gel column 22 ml packed with n-hexane, and n-hexane-ethyl acetate (7: 3)
Elution was carried out with 100 ml and then with 100 ml of n-hexane-ethyl acetate (6: 4).

The eluate was concentrated under reduced pressure, and the yellow oil obtained was dissolved in a small amount of acetonitrile and subjected to high performance liquid chromatography (manufactured by Senshu Kagaku, Senshu Pack (OD
S3301N) 20φ × 300mm), from 20% acetonitrile to 5
Elution was performed with a linear gradient of 0% acetonitrile at a flow rate of 4 ml / min. The active fraction was concentrated under reduced pressure, and then subjected to high performance liquid chromatography (YMC-Pack, A-043 SI manufactured by Yamamura Scientific Research Institute).
L, 20φ × 250mm), n-hexane-chloroform (1: 9)
And eluted at a flow rate of 4 ml / min. The active fraction was concentrated under reduced pressure, dissolved in a small amount of a chloroform-methanol mixture (100: 1), and gradually added with n-hexane to give 3-hydroxymethyl-6-methoxy-8-hydroxy-1H-2. 10 mg of crystals of -benzopyran-1-one were obtained.

[0033]

As described above, the compound (I) of the present invention
Has been shown to significantly reduce rat adjuvant arthritis and collagen-induced arthritis in mice, as well as many immunomodulators, such as rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, periarteritis nodosa and juvenile diabetes. Is expected to be applicable to autoimmune diseases. Moreover, it is particularly excellent as an immunomodulator in terms of toxicity and the like, and can be used effectively and safely.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI C07D 311/76 C07D 311/76 (72) Inventor Rokuro Okamoto 2-18 Hanagi, Fujisawa-shi, Kanagawa Prefecture (72) Inventor Hiroyuki Kumagai Shizuoka 1388 Higashi-Shiji, Numazu City (72) Inventor Masaaki Ishizuka 6-5, Nishiwakacho, Mishima-shi, Shizuoka Prefecture (72) Inventor Tomio Takeuchi 5-1-11 701-A, Higashi-Gotanda, Shinagawa-ku, Tokyo Examiner ▲ Takami Oka (56) reference Patent flat 3-2177 (JP, a) JP Akira 53-71076 (JP, a) JP Akira 60-142976 (JP, a) (58 ) investigated the field (Int.Cl. ⁷ , DB name) C07D 311/76 CA (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] (1) Formula (1) An immunomodulator comprising as an active ingredient 3-hydroxymethyl-6-methoxy-8-hydroxy-1H-2-benzopyran-1-one represented by