JPH05148152A - Immunosuppressant - Google Patents

Abstract

PURPOSE:To obtain an immunosuppresant, containing an extract separated from crushed microbial cell pieces of Bacillus stearothermophilus as an active ingredient and useful for transplanting organs or treating, etc., immunoabnormality diseases. CONSTITUTION:The objective immunosuppressant is obtained by including an extract separated from a crushed microbial cell of Bacillus stearothermophilus which is a medium thermophilic bacterium and a compound expressed by formula I [R1 is formula III (R4 is 14-20C alkyl); R2 and R3 are formula III (R5 is 14-20C alkyl), e.g. 1,3-dipentadecanoin as an active ingredient. The compound which is the active ingredient is obtained by using a method for culturing a strain of the Bacillus stearothermophilus, e.g. NCA-1503 (FERM P-4778) and filtering or centrifuging the culture, etc., or an organic synthetic method for reacting glycerol with an acid halide. The immunosuppresant can be formulated into a tablet, a pellet, a capsule, a suppository, a solution, an emulsion, a suspension, etc.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an excellent immunosuppressive agent, and more particularly to an immunosuppressive agent that can be used for organ transplantation, treatment of immunological disorders and the like.

[0002]

Conventionally, cyclosporine has been used as an immunosuppressant.

[0003]

The above-mentioned drugs that have hitherto been used cannot be said to have sufficient immunosuppressive effects and are not always satisfactory in terms of side effects.

[0004]

[Means for Solving the Problems] The present inventors have conducted extensive studies to find a drug having a high immunosuppressive effect and excellent safety, and it was surprisingly found that Bacillus Bacillus stear
The present invention has been completed by finding the desired activity in crushed cells of othermophilus), pursuing its active ingredient, and clarifying its structure.

That is, the present invention provides an immunosuppressive agent containing an extract of crushed cells of Bacillus stearothermophilus as an active ingredient, and a compound represented by the following general formula as an active ingredient. The subject is an immunosuppressive drug.

[0006]

[Chemical 2]

The present invention will be described in detail below. The active ingredient of the immunosuppressive agent of the present invention is a compound represented by the general formula 2. Examples of the linear or branched alkyl group having 14 to 20 carbon atoms represented by R ₄ and R ₅ include tetradecanoyl, pentadecanoyl, hexadecanoyl, heptadecanoyl,
Octadecanoyl, Nonadecanoyl, Eicosanoyl,
Examples thereof include isotetradecanoyl, isopentadecanoyl, isohexadecanoyl, isoheptadecanoyl, isooctadecanoyl, isononadecanoyl and isoeicosanoyl groups.

Specific compounds include 1,3-dipentadecanoin, 1,3-diisopentadecanoin and 1,3.
-Dihexadecanoin, 1,3-diisohexadecanoin, 1,3-diheptadecanoin, 1,3-diisoheptadecanoin, 1,3-dioctadecanoin, 1,3-
Diisooctadecanoin, 1,3-dinonadecanoin,
1,3-diisononadecanoin, 1,3-diaeicosanoin, 1,3-diisoeicosanoin, 1,3-diheneicosanoin, 1,3-diisoheneicosanoin,
1,2-dipentadecanoin, 1,2-diisopentadecanoin, 1,2-dihexadecanoin, 1,2-diisohexadecanoin, 1,2-diheptadecanoin,
1,2-diisoheptadecanoin, 1,2-dioctadecanoin, 1,2-diisooctadecanoin, 1,2-
Dinonadecanoin, 1,2-diisononadecanoin,
1,2-Dieicosanoin, 1,2-Diisoeicosanoin, 1,2-Dieneeicosanoin, 1,2-Diisoheneicosanoin, Tripentadecanoin, Triisopentadecanoin, Trihexa Decanoin, triisohexadecanoin, triheptadecanoin, triisoheptadecanoin, trioctadecanoin, triisooctadecanoin, trinonadecanoin, triisononadecanoin, trieicosanoin, triisoeico Sanoin,
Trihen eicosanoin, triisohen eicosanoin and the like.

As a method for obtaining a compound which is an active ingredient in the present invention, it is possible to use a method of obtaining a compound from Bacillus stearothermophilus cells as well as an organic synthesis method.

First, a method for extracting and purifying from Bacillus stearothermophilus cells will be described in detail. Bacillus stearothes used in the present invention
rmophilus) is not limited to a particular strain, and specific examples thereof include NCA1503 (Microtechnological Research Institute No. 4778), UK788 (Microtechnical Research Institute No. 23).
No. 73), UK563 (Ministry of Microbiology Research Institute No. 7275), I
FO-12550, IFO-12983, IFO-13
737 and ATCC-7953
No.), ATCC-8005 (Microtech Lab.
No.), ATCC-10149 (Ministry of Industrial Science and Technology, No. 4777)
No.), ATCC-12016, ATCC-12976,
ATCC-12977, ATCC-12978, ATC
C-12979, ATCC-12980, ATCC-1
5951, ATCC-12952, ATCC-2136
5, etc. Among these, NCA-1503
(Microtechnology Research Institute, No. 4778) has the highest immunosuppressive activity in the cells and is most convenient for achieving the purpose.

For culturing these strains, a medium commonly used can be used. Specifically, sugars such as glucose, lactose, starch and molasses can be used as the carbon source. Also, as the nitrogen source, peptone, meat extract,
Inorganic nitrogen sources such as ammonium chloride and ammonium sulfate can be used together with organic nitrogen sources such as yeast extract. In addition, vitamins and minerals may be added if necessary.

As the culture conditions, aerobic culture is adopted,
For example, cells can be obtained by culturing at 58 ° C. for 1 to 12 hours. Furthermore, a continuous culture method of culturing while supplying a fresh medium at a constant rate can also be applied. It goes without saying that the medium and culture conditions can be appropriately selected according to the above conditions.

From the culture solution obtained by culturing as described above,
The cells can be obtained by a conventional method such as filtration or centrifugation. The obtained microbial cells are disrupted by a generally-used microbial cell disruption method such as ultrasonic disruption, grinding with glass beads, French press, self-digestion, etc., and then disrupted cells are obtained by centrifugation or filtration. Ethanol, methanol, from the crushed cells obtained in this way,
Acetone, methylene chloride, chloroform, ethyl acetate,
The active ingredient can be obtained by performing an extraction operation using an organic solvent such as toluene, hexane or benzene. Further, a fraction containing the active ingredient or the active ingredient itself is obtained by an appropriate separation and purification method such as silica gel column chromatography, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, and high performance liquid chromatography, and is used as an immunosuppressant. Can be used.

The immunosuppressive agent of the present invention can also be produced by an organic synthetic method. For example, Biochemistry, Bi
It can be produced by a known method described in Ochemistry, 2, 394 (1963) and the like. That is, glycerol is reacted with acid halide RCl in a solvent such as pyridine,
It can be easily obtained by separating and purifying by silica gel chromatography. Further, the acid halide RCl can be obtained by reacting ROH with thionyl chloride SOCl ₂ .

The compound of the present invention has an excellent immunosuppressive action on mammals and is an excellent immunosuppressant.

The compounds of this invention can be administered to a patient in a variety of forms. For example, tablets, pellets, capsules, suppositories, solutions, emulsions, medicines and the like can be used.
When using these forms, stabilizers, excipients, colorants,
Adjuvants such as fragrances and sweeteners can be used. As the stabilizer, carbon dioxide, nitrogen, sodium hydrogen sulfite, sodium pyrosulfite, ascorbic acid, butylhydroxyanisole, tocopherol, EDTA or the like can be used. As excipients, lactose, starch, crystalline cellulose, sucrose, glucose, mannitol,
Calcium carbonate, calcium phosphate, calcium sulfate,
Sodium chloride, sodium hydrogen carbonate, licorice powder, licorice crude extract, dried yeast, yeast extract, purified lanolin, gentian powder, gentian extract, malt extract,
Glycerin or the like can be used. It can also be administered to a patient by intravenous injection, intramuscular injection, subcutaneous injection, or the like.

The dose is 0.01 per kg body weight.
mg to 100 mg, preferably 0.05 mg to 50 m
g, more preferably 0.1 mg to 10 mg is suitable, but it goes without saying that the optimum dose is appropriately selected depending on the patient's symptoms, the degree of progression of the medical condition, and the like.

[0018]

EXAMPLES The present invention will be described in more detail below with reference to examples. In the examples,% represents W / V%. Example 1 Bacillus stearot
hermophilus) NCA-1503
No. 8) glucose 0.35%, yeast extract 0.3%,
2.2 liters of a culture medium inoculated into a liquid medium composed of 0.2% peptone, 0.04% potassium phosphate, 0.04% disodium phosphate and 0.1% magnesium sulfate and precultured at 58 ° C. for 4 hours Was transferred to a 30-liter jar fermenter containing 18 liters of the same medium and cultured at 58 ° C. for 3.5 hours.

The culture solution was centrifuged and the obtained bacterial cells were added to 20 mM.
It was self-digested by stirring in a phosphate buffer (pH 8) -2 mM sodium ethylenediaminetetraacetate at 40 ° C. for 2 hours. Then, centrifugation was performed to obtain a precipitate. This precipitate (500 g) was extracted twice with 4 l of hot ethanol (10 minutes, 70 ° C). This extracted fraction was dried under reduced pressure, dissolved in 1 liter of water, and extracted 4 times with 500 ml of methylene chloride. This was subjected to silica gel column chromatography, and chloroform / methanol (100 /
Elution was performed in 1) to obtain an active fraction KM.

The active fraction KM thus obtained was further subjected to silica gel column chromatography and eluted with n-hexane / ethyl acetate (2/1) to obtain the active fraction KM-.
I got 73. The yield of the active fraction KM-73 was 91.1 mg from 500 g of bacterial cell fragment precipitate.

To determine the structure of this product, ¹³ C-NM
When R, ¹ H-NMR and infrared absorption spectrum were measured, 1,4 having a structure in which R ₄ and R ₅ in the general formula shown in Chemical formula ₂ are isoheptadecanoyl groups and R ₂ is a hydrogen atom -It was found to be dioctadecanoin.

FIG. 1 shows the ¹ H-NMR analysis result, and FIG. 2 shows the infrared absorption spectrum analysis result.

Example 2 Using the active ingredient obtained in Example 1, a mouse mixed lymphocyte reaction (MLR) was carried out to examine the immunosuppressive activity of the compound of the present invention. Preparation of splenocyte suspension: C57BL / 6 mice and
The spleen was removed from the BALB / C mouse, immersed in about 5 ml of Hanks' solution (Nissui Pharmaceutical, HBSS), and splenocytes were extruded from the spleen with a slide glass to obtain a uniform cell suspension. This cell suspension was filtered through a nylon mesh and then centrifuged to remove red blood cells using 17 mM Tris-144 mM ammonium chloride buffer (pH 7.2). After washing the splenocytes with Hanks' solution, the number of cells was adjusted and used.

Preparation of stimulator cells: BALB / C splenocytes 5 × 1
RPMI 1640 (Nissui Pharmaceutical Co., Ltd.) to achieve 0 ⁷ / ml
Then, 100 μl of mitomycin C (manufactured by Wako Pure Chemical Industries, Ltd.) having a concentration of 500 μg / ml was added, and the mixture was left at 37 ° C. for 30 minutes. The cells were washed three times with a Hank's solution by centrifugation, prepared using a medium (GIBCO, RPMI1640) so that the number of cells would be 6.7 × 10 ⁶ / ml, and used as stimulator cells.

Preparation of Responsive Cells: C57BL / 6 splenocytes were used in 6.
7x10 medium such that the ⁶ / ml concentration (GIBCO
Company, RPMI1640) and used as responder cells.

Preparation of sample: The sample is dissolved in ethanol,
It was adjusted so that the final ethanol concentration was 0.4%.

Mixed lymphocyte reaction: 50 μl of a sample was placed in each well of 75 μl of the above-mentioned two types of cell suspensions in a well of a flat bottom 96-well microplate. After culturing for 4 days in a carbon dioxide incubator adjusted to 37 ° C., 5% carbon dioxide-95% air, [ ³ H] -thymidine (Amer
50 μl of ican Radiolabelled Chemicals Inc.) was added, and the cells were further cultured in a carbon dioxide gas incubator for 16 hours. After completion of the culturing, the cell harvester cells were collected on a glass filter paper, and the [ ³ H] -thymidine incorporation amount was measured by a liquid scintillation counter. The inhibition rate of the amount of thymidine uptake for the sample-free group was determined. Cyclosporine was used as a comparative sample.

FIG. 3 shows the results of examining the effects of the KM-73 and cyclosporine, which are the products of the present invention, on the mixed lymphocyte reaction in mice. Since KM-73 suppresses the mixed lymphocyte reaction at a lower concentration than cyclosporin, it can be seen that its effect as an immunosuppressant is excellent.

Example 3 The procedure of Example 2 was repeated, except that the sample was added 48 hours after the start of the mixed lymphocyte reaction.

FIG. 4 shows the results of mixed lymphocyte reaction.
KM-73 compared to conventional drug cyclosporine
It can be seen that the immunosuppressive effect is remarkable when the compound is added after the mixed lymphocyte reaction is started, and the immunosuppressive effect is remarkable.

Example 4 KM-73 5 mg Glucose 100 mg Physiological saline 10 ml The above mixed solution was filtered through a membrane filter and then sterilized again, and the filtered solution was aseptically dispensed into a vial.
After filling with nitrogen gas, it was sealed and used as an intravenous injection drug.

Example 5 KM-73 50 g Lactose 935 g Magnesium stearate 15 g The above ingredients were weighed and mixed uniformly, and 200 mg each of hard gelatin capsules were filled with the mixed powder to prepare capsules.

[0033]

INDUSTRIAL APPLICABILITY The immunosuppressive agent of the present invention has an excellent immunosuppressive effect and can be effectively used as a therapeutic agent for organ transplantation and immunological disorders.

[Brief description of drawings]

FIG. 1 is the ¹ H-NMR analysis result of a purified preparation of the active ingredient of the present invention.

FIG. 2 is an infrared absorption spectrum analysis result of a purified preparation of the active ingredient of the present invention.

FIG. 3 is an explanatory view showing the effect of the active ingredient of the present invention on the mixed lymphocyte reaction.

FIG. 4 is an explanatory view showing the effect when the active ingredient of the present invention is added after the start of the mixed lymphocyte reaction.

Claims (2)

[Claims] 1. Bacillus stearothermophilus (Ba
cillus stearothermophilus) An immunosuppressive agent containing an extract of crushed bacterial cells as an active ingredient. 2. An immunosuppressive agent comprising a compound represented by the following general formula as an active ingredient. [Chemical 1]