JPH07277970A - New immunosuppressive agent - Google Patents

Abstract

PURPOSE:To provide an immunosuppressive agent containing demethylpyronetin as an active component and useful for the treatment of rejection to the transplantation of organ or tissue, graft versus host reaction caused by marrow transplantation and various autoimmune diseases. CONSTITUTION:This agent contains 0.1-100wt.% of the compound of formula, i.e., (5R*, 6R*)-5-ethyl-5,6-dihydro-6-[(E)-(2R*, 3S*,4R*,5S*)-2,4-dihydroxy-3,5- dimethyl-7-nonenyl]-2H-pyran-2-one (demethylpyronetin) or its salt as an active component. The daily dose of the component is 5-500mg/kg for oral administration and 1-100mg/kg for parenteral administration. The agent does not cause grave side effects such as hematopoietic disorder and gastric ulcer, cataract, etc., noticeable in the case of using steroid hormones.

Description

Detailed Description of the Invention

[0001]

The present invention relates to the following formula (1)

[0002]

[Chemical 2]

Demethylpyronetin (NK10 represented by
958P) as an active ingredient.

[0004]

2. Description of the Related Art Alkylating agents have been used as immunosuppressants.
Antimetabolites, antibiotics, steroids, antifolates, plant alkaides, etc. are known. On the other hand, demethylpyronetin (NK10958P) is produced by microorganisms, and is known to have agricultural and horticultural use, herbicidal action, plant growth regulating action, antibacterial activity, etc. (JP-A-5-1481).
17 publication).

[0005]

Among the conventional immunosuppressive agents, steroids are said to exhibit immunosuppression due to anti-inflammatory action, lympholytic action, etc., and have a wide variety of actions.
It is well known that various side effects are involved.

It is known that other immunosuppressive agents belong to so-called cytotoxic substances, and in particular, many of them act on the nucleic acid synthesis system and easily cause serious side effects in organs such as hematopoietic organs. There is a demand for a drug that selectively acts on immunocompetent cells such as lymphocytes and has minimal side effects other than immunosuppressive effects.

[0007]

Therefore, as a result of various investigations, the inventors of the present invention obtained the formula (1)

[0008]

[Chemical 3]

Dimethylpyronetin (NK10 represented by
958P) was found to have an immunosuppressive effect.
The present invention has been completed based on the above findings.

That is, the present invention relates to demethylpyronetin (NK
10958P) or a pharmacologically acceptable salt thereof as an active ingredient.

Demethylpyronetin (NK10958P) represented by the formula (1) in the present invention is disclosed in JP-A-5-14.
As described in Japanese Patent No. 8117, it is a known compound produced by Streptomyces SP. NK10958 strain (Microtechnology Research Institute No. 12317). (Hereinafter, the compound represented by the above formula (1) of the present invention is referred to as "the present compound".)

This compound is the compound described in the above-mentioned JP-A-5-148117.
According to the method described in Japanese Patent Publication No. NK10958,
It can be advantageously produced using the strain.

Further, in general, the radiobacterium is liable to be naturally or artificially mutated in its properties, and the antibiotic of the present invention can be produced by using a mutant strain of NK10958 strain, and is not limited to the above. Alternatively, it can be produced by using various known bacteria belonging to the genus Streptomyces.

In order to produce the present compound, first, the strain is aerobically cultivated in a medium containing nutrients that can be utilized by the radiant bacterium. As the nutrient source, known ones which have been conventionally used for culturing a radiant bacterium can be used.For example, as the carbon source, glucose, fructose, glycerin, sucrose, dextrin, galactose, an organic acid or the like alone or It can be used in combination.

As the inorganic and organic nitrogen sources, ammonium chloride, ammonium sulfate, urea, ammonium nitrate,
Sodium nitrate, peptone, meat extract, yeast extract, dry yeast, corn steep liquor, soybean flour, cottonseed oil residue, casamino acid, bactsonton, soluble vegetable protein, oatmeal and the like can be used alone or in combination.

In addition, inorganic salts such as sodium chloride, calcium carbonate, magnesium sulfate, copper sulfate, iron sulfate, zinc sulfate, manganese chloride, and phosphate can be added, if necessary, and organic substances such as amino acids, vitamins and nucleic acids. It is possible to appropriately add a substance or an inorganic substance. As the culturing method, the liquid culturing method, particularly the deep agitation culturing method is most suitable.

It is desirable to carry out the culture at a culture temperature of 20 to 45 ° C. and a slightly acidic or slightly alkaline pH. In liquid culture, when the culture is generally performed for 1 to 5 days, the present compound is produced and accumulated in the culture medium. When the production amount in the culture solution reaches the maximum, the culture is stopped, and the target product is purified and isolated from the culture solution obtained by filtering the bacterial cells.

For purification and isolation of the substance from the culture filtrate, a separation and purification method generally used for isolating microbial metabolites from the culture medium is used. For example, the culture is separated into a filtrate and a precipitate by a filtration method. The filtrate is adsorbed on the porous polymer resin and eluted with acetone. The precipitate is extracted with acetone.

The obtained acetone solution is further dissolved in a general organic solvent immiscible with water and concentrated under reduced pressure to obtain an oily substance containing the present compound. The crude substance is further purified by a known method usually used for lipophilic purification, that is, chromatography using silica gel, or recrystallization method alone or in combination.

Silica gel is used as a suitable example for purification,
A column chromatography method using n-hexane ethyl acetate or n-hexane-acetone as an eluent can be mentioned. A colorless crystal of the present compound can be obtained by concentrating and drying the active fraction obtained by these methods.

The present compound forms a salt with an acid. The acid for forming the salt may be any pharmacologically acceptable acid, and for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and the like are preferable.

From the X-ray crystallographic analysis and the like, the three-dimensional structure of the present compound obtained as described above is (5R ^* , 6R ^* )-5-
Ethyl-5,6-dihydro-6-[(E)-(2R ^* ,
3S ^* , 4R ^* , 5S ^* )-2,4-dihydroxy-
3,5-Dimethyl-7-nonenyl] -2H-pyran-2
-Confirmed to be on. In this compound, the hydroxyl groups at the 2- and 4-positions of the side chain nonenyl group may form an ester with an acid such as carboxylic acid or phosphoric acid, and further may form an ether.

When this compound is used as an immunosuppressant, it is administered alone or in admixture with an excipient or carrier as an injection, oral preparation, suppository or the like. As the excipient and carrier, those which are pharmaceutically acceptable are selected,
The type and composition thereof depend on the administration route and administration method. For example, water, alcohol or soybean oil, peanut oil, sesame oil, mineral oil or other animal or vegetable oil as a liquid carrier,
Or maltose as a solid carrier in which synthetic oil is used,
Sugars such as sucrose, amino acids, cellulose derivatives such as hydroxypropyl cellulose, and organic acid salts such as magnesium stearate are used.

In the case of injections, physiological saline, various buffer solutions, sugar solutions such as glucose, inositol and mannitol, ethylene glycol, propylene glycol, etc.
Glycols such as polyethylene glycol are desirable.
Inositol, mannitol, glucose, mannose, maltose, sugars such as sucrose, and a freeze-dried preparation together with excipients such as amino acids such as phenylalanine, a suitable solvent for injection at the time of administration, such as sterile water, It can also be administered by dissolving it in a liquid for intravenous administration such as physiological saline, glucose solution, electrolyte solution and amino acid solution.

The content of the present compound in the preparation varies depending on the preparation, but is usually 0.1 to 100% by weight, preferably 1
Is about 98% by weight. For example, in the case of an injectable solution, it is preferable to contain the active ingredient in an amount of usually 0.1 to 30% by weight, preferably 1 to 10% by weight. In the case of oral administration, it is used in the form of tablets, capsules, powders, granules, liquids and dry syrups together with the above solid or liquid carriers. Capsules, tablets, granules and powders generally contain 2-100% by weight, preferably 5-98% by weight, of the active ingredient.

The dose is determined according to the age, body weight, condition of the patient, purpose of treatment and the like. Generally, the dose is 1 to 100 mg / kg · day for parenteral administration and 5 to 500 mg / day for oral administration.
kg / day.

(Action) This compound exerts an inhibitory action on the function of mouse lymphocytes which are immunocompetent cells. That is, Waithe
(Waithe et al., Handbook of Experimen
tal Immunol-ogy p. 26, 1, 1978), the effect on the lymphocyte blast transformation reaction was examined.
(Concanavalin A) -stimulated blastogenesis of T lymphocytes and LPS (lipopolysaccharide) -stimulated B lymphocytes were remarkably suppressed. The pharmacological action of the present compound described above will be specifically described with reference to test examples.

Test Example 1. Suppression of T lymphocyte blastogenic reaction by Con A 2x splenocytes of BALB / C mice were placed on a microplate.
Dispense to 10 ⁵ cells / 0.2 ml / well, add each concentration of test compound to each well other than the control group, and further add Con A to all wells at 5 μg / ml. The cell suspension was cultured at 37 ° C. in a 5% carbon dioxide incubator for 72 hours.

In the lymphocyte blastogenesis reaction, ³ H-thymidine was added at 1 μCi / well 8 hours after the end of the culture, and the uptake into the cultured cells was measured by a liquid scintillation counter. The uptake count when only Con A was added is Adp
The count when m, Con A and the drug were added was taken as Bdpm, and the numerical value of (1-Bdpm / Adpm) × 100 was taken as the inhibitory rate of each drug against aging. The results are shown in Table 1.

[0030]

[Table 1]

As is clear from the above table, this compound strongly suppressed the T lymphocyte blastogenic reaction.

Test Example 2. Suppression of B lymphocyte blastogenic reaction by LPS (lipopolysaccharide) In accordance with the method of Test Example 1 (however, instead of Con A, 1
The amount of ³ H-thymidine incorporated into the immature B cells was measured. The inhibition rate by the test compound was similarly determined. As shown in Table 2, this compound markedly suppressed the B lymphocyte blastogenic reaction induced by LPS.

[0033]

[Table 2]

The above results show that the present compound suppresses the functions of B lymphocytes and T lymphocytes. Since this suppressive action means suppression of humoral immunity and cellular immunity respectively, suppression of rejection in organ transplantation or skin transplantation, which is considered to be caused by its abnormal increase, and various autoimmunity are considered to be the main causes. The utility of the present compounds for the treatment of immune diseases such as multiple sclerosis, hemolytic anemia, type I diabetes, myasthenia gravis, Hashimoto's thyroiditis, Betiet's syndrome rheumatism or allergic diseases is strongly suggested.

Since this compound may have a different mechanism of action from conventional immunosuppressive agents, hematopoietic disorders and the like commonly observed in inhibitors belonging to cytotoxic substances, and gastric ulcers and cataracts and the like observed with steroid hormones. It is considered that there are no serious side effects.

As is clear from the above description, the present compound exhibits an excellent immunosuppressive action such as a function-suppressing action on T lymphocytes and B lymphocytes and an antibody-producing ability against sheep erythrocytes. The mechanism of action is considered to be different from that of the suppressive agent, and is expected as an immunosuppressive agent with few side effects.

[0037]

EXAMPLES The present invention will be specifically described below with reference to examples.

Example 1 Injectable solution Distilled water was added to 30 parts by weight of the hydrochloride of the present compound to make a total amount of 2000 parts, which was dissolved and then Millipore Filter GS.
Sterilize and filter using the type. 2 g of this filtrate is 10 ml
Was freeze-dried to obtain a lyophilized injection containing 30 mg of the hydrochloride of the present compound in 1 vial.

Example 2 Granules 50 parts by weight of hydrochloride of the present compound, 600 parts of lactose, 330 parts of crystalline cellulose and 20 parts of hydroxypropyl cellulose.
The parts were mixed well, compressed using a roll type compressor (roller compactor; registered trademark), crushed and sieved so as to be between 16 mesh and 60 mesh to give granules.

Example 3 Tablets 30 parts by weight of the hydrochloride of the present compound, 120 parts of crystalline lactose, 147 parts of crystalline cellulose and 3 parts of magnesium stearate were tabletted by a V-type mixer to give tablets of 300 mg each.

[0041]

INDUSTRIAL APPLICABILITY Since the compound of the present invention has an immunosuppressive action, it is useful for the treatment of rejection of organ or tissue transplantation, graft-versus-host reaction caused by bone marrow transplantation, and various autoimmune diseases. .

Claims (2)

[Claims] 1. The following formula (1): A novel immunosuppressive agent comprising a compound represented by: or a pharmacologically acceptable salt thereof as an active ingredient. 2. A compound of formula (1) is (5R ^* , 6R ^* ) 5.
-Ethyl-5,6-dihydro-6-[(E)-(2
R ^* , 3S ^* , 4R ^* , 5S ^* )-2,4-dihydroxy-3,5-dimethyl-7-nonenyl] -2H-pyran-2one or a pharmacologically acceptable salt thereof.
Immunosuppressant.