JPH0578307B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a new method for producing an immunosuppressive substance, particularly myriocin, using microorganisms. [Prior Art] Conventionally, it has been known that microbial-derived immunosuppressive substances can be produced by culturing immunosuppressive substance-producing strains belonging to the genus Cylindrocapon, Tolypocladium, or Fusarium. Are known. In particular, cyclosporine produced from the genus Tolypocladium is widely used to prevent rejection during organ transplants. [Purpose of the Invention] In view of the above circumstances, the present inventors searched for microorganisms that produce immunosuppressive substances and found that immunosuppressive substances were detected from microorganisms belonging to a certain genus that was not known to produce immunosuppressive substances. We discovered a substance-producing bacterium and arrived at the present invention. [Structure of the Invention] That is, the gist of the present invention is based on Isaria.
The present invention relates to a method for producing an immunosuppressive substance, which comprises culturing an immunosuppressive substance-producing bacterium belonging to the genus and collecting the immunosuppressive substance from the culture. The present invention will be explained in detail below. The microorganism used in the method of the present invention belongs to the genus Isaria and is an immunosuppressant-producing bacterium that has the ability to produce a sufficient amount of an immunosuppressant in culture.
Examples of such strains include sinclairii, which belongs to the genus Isaria, and is included in the American Type Culture Collection.
It has been deposited as Isaria sinclairii ATCC No. 24400. The immunosuppressive substance-producing bacteria used in the present invention are not limited to these strains, and of course include mutant strains that have been modified by artificial mutation means such as commonly used ultraviolet rays, high-frequency radiation, and chemicals. The immunosuppressant-producing bacteria used in the present invention can be cultured in various culture media containing common nutrient sources for molds.
For example, carbon sources include glucose, starch, glycerin, sugar syrup, dextrin, molasses, maltose, etc., and nitrogen sources include cornstarch liquor, peptone, yeast extract, potato broth, meat juice, soy flour, wheat germ, potassium nitrate, etc.
Examples include sodium nitrate, ammonium sulfate, amino acids, and other common inorganic salts and additives commonly used in culture such as organic and inorganic substances that aid the growth of bacteria and promote the production of immunosuppressive substances, and antifoaming agents. can be added. Although the culture method is not particularly limited, an aerobic deep culture method is suitable. A suitable culture temperature is 20 to 35°C, preferably 25 to 30°C. The immunosuppressive substance produced in the culture by the method of the present invention can be extracted from the culture by appropriately combining commonly used operations such as extraction and adsorption as necessary. For example, insoluble substances such as bacterial cells are separated from the culture solution by filtration, centrifugation, or the like, and the culture filtrate is passed through Amberlite XAD-2 to adsorb the immunosuppressive substance. The immunosuppressive substance thus obtained may be further treated with, for example,
A highly purified product of the immunosuppressive substance can be obtained by elution with methanol and further subjecting the eluate to reverse phase chromatography and fractionation. The highly purified immunosuppressive substance obtained in this way is derived from Myriococcum albomyces.
Myriocin, also known as Thermozymocidin, which is also produced by bacteria such as Albomyces and Mycelia sterilia [US Patent No. 3928572
It was inferred from NMR, ultraviolet absorption spectrum, infrared absorption spectrum, mass analysis, etc. This substance also has strong immunosuppressive activity. [Effect] The immunosuppressive substance produced by the present invention exhibits an excellent immunosuppressive effect on humans, cows, horses, dogs,
For mammals such as mice and rats, for example, an inhibitor of rejection during organ or bone marrow transplantation, or a prophylactic or therapeutic agent for autoimmune diseases such as lupus erythematosus, rheumatoid arthritis, allergies, or diseases based on abnormal lymphocyte proliferation. or as a reagent in medicine and pharmacy. When the immunosuppressive substance of the present invention is used as the above-mentioned medicine, it can be mixed with carriers, excipients, diluents, etc., formulated into powders, capsules, tablets, injections, etc., and administered to patients. Alternatively, it may be freeze-dried. The dosage may vary depending on the disease, symptoms, body weight, sex, age, etc., but for example, to suppress rejection in kidney transplantation, it is usually administered per day for an adult.
It is administered in doses of 0.1 to 10 mg (potency) in one to several divided doses a day. [Examples] The present invention will be described in more detail with reference to Examples below, but the present invention is not limited in any way by the Examples unless the gist of the invention is exceeded. The activity of the immunosuppressive substance was measured by the following method. As a method for measuring the activity of immunosuppressive substances, mice,
Although various immune reactions using rat or human lymphocytes can be used, for example, the immunosuppressive substance of the present invention can be used in a mixed reaction (allogeneic MLR) of mouse, rat, and human allogeneic lymphocytes. Can be measured with good sensitivity. What is homogeneous MLR?
A lymphocyte rejuvenation reaction induced by mixed culture of lymphocytes from two individuals of the same species but with different major histocompatibility antigens, such as splenocytes, lymph node cells, and peripheral blood lymphocytes. be. This allogeneic MLR is a phenomenon that reflects and is induced by differences in major histocompatibility antigens between lymphocyte donors. unacceptable. Allogeneic MLR, for example, is a widely used method for donor-recipient selection in organ transplants. Normally, when performing allogeneic MLR, one lymphocyte is used as a stimulator cell after its division and proliferation is inhibited by X-ray irradiation or mitomycin C treatment, and the other lymphocyte (reactive cell) is used as a stimulator cell. How to measure the rejuvenation response of one
way-MLR) can be used. Furthermore, the activity of the immunosuppressant of the present invention is similar to that of the same species.
It can also be measured as the activity of suppressing the induction of cytotoxic T cells with major histocompatibility antigen-restricted properties induced during MLR. Furthermore, the activity of the immunosuppressive substance of the present invention is similar to that of the same species.
In addition to MLR, the activity of suppressing the lymphocyte blastogenesis induced by stimulation with various mitogens (concanavalin A, phytohemagglutinin, porkweed mitogen, etc.), or T
Cytokines (interleukins 1, 2, 3, 4, 5,
It can also be evaluated as the activity of suppressing lymphocyte division/proliferation response or function induction activity induced by (6, etc.). Furthermore, it is possible to evaluate the activity of suppressing the production of these cytokines from T cells, macrophages, etc. Furthermore, the immunosuppressive substance of the present invention can be administered intraperitoneally, orally, intravenously, or intradermally to mice, etc., to induce anti-spleen cells of mice that have been immunized with xenogeneic red blood cells. It can also be evaluated as the activity of suppressing the induction of plasma cells that produce xeno-erythrocyte antibodies, the graft-versus-borrower reaction induced by skin transplantation of allogeneic mice, or the activity of suppressing delayed-type allergies, adjuvant arthritis, etc. . It is also a mouse model for autoimmune diseases.
MRL/pr mouse, NZB/WF ₁ mouse, BXSB
By administering the immunosuppressive substance of the present invention to mice and the like, it can be evaluated, for example, as suppressive activity against anti-DNA antibody production, rheumatoid factor production, nephritis, abnormal lymphocyte proliferation, etc., or as a survival effect. [Example] Example 1 (Jear culture of Sinclairy strain) GPY medium (30 g of glycose, 5 g of peptone, 3 g of yeast extract, 0.3 g of KH ₂ PO ₄ , K ₂ per portion)
HPO ₄ 0.3g, MgSO ₄・7H ₂ O 0.3g, PH5.5)
Dispense 100 ml into two 500 ml long shake flasks, sterilize in an autoclave at 121°C for 20 minutes, and add approximately 1 cm ² of Isaria Sinclairi ATCC No. 24400 mycelium grown on potato dextrose agar to each flask. and cultured with reciprocating shaking (145 rpm, shaking width 8 cm) at 25°C for 6 days.
Using the obtained culture solution as a seed mother, use the above GPY medium 5.
was inoculated into a 10-volume jar of fermenter, and cultured with aeration at 25°C for 10 days (1VVM,
300rpm). Example 2 (Collection of immunosuppressive substance from culture solution of Bacillus cinquerilii) Bacterial cells and insoluble matter were removed by filtration from culture solution 4.5 obtained in Example 1 to obtain culture filtrate 4.0. The obtained culture filtrate was injected into Amberlite XAD-
2 (φ40 mm x 750 mm) to adsorb the immunosuppressive substance. Further, water 4 was passed therethrough for washing. Thereafter, methanol 6 was passed through the tube to elute the immunosuppressive substance. This was concentrated under reduced pressure, dissolved in 200 ml of ethyl acetate, and extracted three times with 200 ml of water using a funnel. The water extract and ethyl acetate were each concentrated under reduced pressure and then lyophilized to produce 2.23 g of the immunosuppressant and
0.34g each was obtained. Example 3 (Collection of myriocin) 2.23 g of the immunosuppressive substance obtained by freeze-drying the water extract obtained in Example 2 was dissolved in 5 ml of water,
Reversed phase chromatography (ODS DM-1020T manufactured by Fuji Davison Chemical) column (φ30mm x
h85mm). Elution was started with water and fractionated by elution while gradually increasing the methanol concentration. The 70% methanol eluate was concentrated to dryness under reduced pressure, then dissolved in a small amount of hot methanol, and allowed to cool to precipitate myliosin crystals. The precipitated crystals were dissolved again in hot methanol and recrystallized to obtain 40 mg of pure myliocin. [Experimental Examples] Experimental Example 1 (Measurement of the immunosuppressive effect of the collected immunosuppressive substance) The activity of the immunosuppressive substance of the present invention was measured using a mouse allogeneic lymphocyte mixed reaction (hereinafter sometimes referred to as MLR). I did it. Mouse allogeneic MLR
BALB/c mice (H-2 ^d ) were used as reactive cells.
C57BL/6 (H- ^2b ) splenocytes were used as stimulator cells.
The experiment was carried out by using mouse splenocytes treated with mitomycin C and culturing them in equal proportions. The reaction cells were prepared by the following method. The spleen was removed from a 5- to 6-week-old male BALB/c mouse and treated with heat-inactivated fetal bovine serum (hereinafter referred to as FCS).
It is also sometimes called. ) was added at 5%
RPM11640 medium (kanamycin sulfate 60 μg/ml,
A single cell suspension of splenocytes was obtained using 2 mM L-glutamine, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate (HEPES), and 0.1% sodium bicarbonate. After the hemolysis treatment ^, 10 ⁷
cells/ml and used as a reaction cell suspension. The stimulated cells were prepared as follows. 5
The spleen was removed from a ~6 week old male C57BL/6 mouse, and a single cell suspension of splenocytes was obtained using RPM11640 medium. After hemolysis, treatment was performed with 40 μg/ml mitomycin C at 37° C. for 60 minutes. After three washes, 10 ^-4 M2-mercaptoethanol and 20
Using RPM11640 medium containing %FCS, 10 ⁷ cells/
ml and used as a stimulated cell suspension. Reactive cell suspension prepared by the method described above
50μ, stimulated cell suspension 50μ and test body fluid
100μ was added to a 96-well microtest plate, and cultured for 4 days at 37°C under 5% carbon dioxide gas. As a method for measuring lymphocyte blastogenesis,
MTT([3-(4,5-dimethylazole-2-
A dye quantitative method using 2,5-diphenyltetrazolium bromide]) and a method using ³ H-thymidine incorporation as an indicator were used. 1. Dye determination method using MTT After the completion of culture, remove 100μ of supernatant from each well,
20μ of 5mg/ml MTT solution was added to each well and incubated at 37°C for 4 hours. Then, add 100μ of 0.01N hydrochloric acid solution containing 10% sodium dodecyl sulfate and incubate at 37°C overnight to dissolve the formed purple formazan crystals and analyze using a microplate spectrophotometer.
Measure the absorbance at 550 nm and measure the absorbance at 550 nm.
It was used as an indicator of MLR lymphocyte blastogenesis. Suppression of mouse allogeneic MLR was evaluated by calculating the suppression rate using the following formula. Suppression rate (%) = [1 - (absorbance of MLR with analyte added) - (absorbance of only reactive cells) / (ML without analyte added)
Absorbance of R) - (absorbance of only reacting cells)] × 100 Method using 2 ³ H thymidine incorporation as an indicator After culturing, add 0.5 μCi/well of ³ H thymidine, and after culturing for 4 hours, remove the cells using a cell harvester. were collected, and the radioactivity incorporated into the cells was measured using a liquid scintillation counter, which was used as an indicator of lymphocyte development in mouse allogeneic MLR. Suppression of mouse allogeneic MLR was evaluated by calculating the suppression rate using the following formula. Inhibition rate (%) = [1 - (cpm of MLR with analyte added)
- (cpm of reactive cells only) / (cpm of MLR without analyte added)
) - (cpm of reactive cells only) ] × 100 After suspending the immunosuppressive substance of the present invention in methanol,
It was diluted with RPM11640 medium and used. Note that methanol was used at a final concentration of 0.1% or less, and in this case, no effect was observed on the homogeneous MLR. Regarding the immunosuppressive substance of the present invention (water extract fraction and ethyl acetate extract fraction), from 10 μg/ml to 10 ^-4
As a result of measuring the inhibitory activity of lymphocyte blastogenesis in mouse allogeneic MLR at a final concentration in the range of μg/ml, as shown in Table 1, the immunosuppressive substance of the present invention (ethyl acetate extraction) was 50% MLR
The inhibitory concentration (IC ₅₀ ) is 1.6×10 ^-3 μg/ml,
The IC ₅₀ of the water-extracted immunosuppressant is 2.5×10 ^-1 μg/ml
It became clear that. On the other hand, these immunosuppressive substances showed no cytotoxicity against mouse L929 cells, etc., even at a concentration of 10 μg/ml (IC ₅₀ is 50 μg/ml or higher).

[Table] Experimental Example 2 (Measurement of the immunosuppressive effect of myliocin) The immunosuppressive effect of myliocin was measured in the same manner as in Example 1 using the method using ^3H -thymidine uptake as an indicator, and the results are shown in Table 2. Ta. Note that myriocin was suspended in methanol. In addition, the final concentration of methanol is used at 0.05% or less,
In this case, no effect was observed on allogeneic MLR. IC ₅₀ of myriocin against mouse homologous MLR
The value is less than 1/100 that of cyclosporin A.

[Table] Experimental Example 3: Suppressing effect on mouse splenocyte rejuvenation response induced by mitogen stimulation) Testing of the effect on mouse splenocyte rejuvenation response induced by phytohemagglutinin (PHA) or porkweed mitogene (PWM) stimulation was as follows: I did it in the following way. Spleens were removed from male BALB/c mice aged 5 to 8 weeks, and 5% heat-inactivated fetal bovine serum was added.
A single cell suspension of splenocytes was obtained using RPM11640 medium. After hemolysis treatment, containing 10 ^-4 M2-mercaptoethanol and 20% heat-inactivated fetal bovine serum.
Adjust to 5 x ¹⁰⁶ cells/ml using RPMI1640 medium.
Added PHA or PWM. This cell suspension
100μ of the test solution was added to each well of a 96-well microtest plate in which 100μ of the test solution had been previously placed (the number of mouse splenocytes was 5×10 ⁵ cells/well). After culturing at 37° C. for 72 hours under 5% carbon dioxide gas conditions, 0.5 μCi/well of ³ H-thymidine was added, and the cells were cultured for an additional 4 hours under the same conditions. After the culture was completed, the cells were collected using a cell harvester, and the radioactivity incorporated into the cells was measured using liquid scintillation countermeasures, which was used as an indicator of the mouse splenocyte rejuvenation reaction. The results are shown in Table 3.

[Table] As is clear from Table 3, the immunosuppressive substance of the present invention strongly suppressed ³ H-thymidine uptake induced by PHA or PWM, compared to the control not containing the same substance. Experimental Example 4 (IL2-dependent mouse cell line induced by interleukin 2 (IL2), CTLL-
2. Inhibitory effect on ^3H -thymidine uptake) IL2-dependent mouse cell line CTLL-2 was cultured at 2 x ¹⁰ cells/ml in RPMI1640 medium containing 30% fetal bovine serum.
It was prepared as follows. 50μ of this cell suspension and concanavalin A-stimulated rat splenocyte culture supernatant containing IL2.
50μ and 100μ of test solution were added in advance.
was added to each well of a 96-well microtest plate. 20, 44 and 68 at 37℃ and 5% carbon dioxide
After culturing for an hour, 0.5 μCi/well of ³ H-thymidine was added, and the cells were further cultured for 4 hours under the same conditions. After culturing, collect the cells using a monkey harvester,
Radioactivity taken into the cells was measured using a liquid scintillation counter. The results are shown in Table 4.

【table】

[Table] As is clear from Table 4, the immunosuppressive substance of the present invention suppresses CTLL-2 ^cells induced by IL2.
The increase in H-thymidine uptake was strongly suppressed. Experimental Example 5 (Suppressive effect on IL2 production in mouse allogeneic lymphocyte mixed culture (MLC)) Mouse allogeneic MLC was performed as follows. That is, 0.5 ml each of the reactive cell suspension and stimulated cell suspension prepared in the same manner as in Experimental Example 1,
It was added to a 24-well multi-dish into which 1 ml of the test solution had been previously placed, and cultured for 3 days at 37°C and 5% carbon dioxide. After completion of the culture, the supernatant was collected and used as a supernatant of mouse allogeneic MLC. IL2 activity in the supernatant of mouse allogeneic MLC was measured as follows. Specifically, the IL2-dependent mouse cell line CTLL-2 was prepared at ¹⁰⁵ cells/ml in RPMI1640 medium containing 30% heat-inactivated fetal bovine serum, and 100μ of the above MLC supernatant was added in advance96.
100μ in each well of the hole micro test plate
Added one by one. After culturing at 37° C. for 20 hours under 5% carbon dioxide gas conditions, 0.5 μCi/well of ³ H-thymidine was added, and the cells were further cultured under the same conditions for 4 hours. After the culture was completed, the cells were collected using a cell harvester, and the radioactivity incorporated into the cells was measured using a liquid scintillation counter, which was used as an index of IL2 activity. The results are shown in Table 5.

[Table] As shown in Table 5, it was suggested that the immunosuppressive substance of the present invention suppresses IL2 production in mouse allogeneic MLC. Experimental Example 6 (Suppressive effect on allogeneic cytotoxic T cells induced by mouse allogeneic lymphocyte mixed culture (MLC)) BALB/
c Mouse (H- ^2d ) splenocyte suspension (2 x 10 ⁷ cells/
ml) 0.5 ml and mitomycin C treated
C57BL/6 mouse (H- _2b ) splenocyte suspension (2x
¹⁰⁷ cells/ml) and 1.0 ml of the test substance were added to a 24-well multi-dish and cultured for 6 days at 37°C under 5% carbon dioxide gas. After culturing, collect the cells by centrifugation and 10%
5×10 ⁶ to 6.25× in RPMI1640 medium containing FCS
The cells were adjusted to 10 ⁵ cells/ml and used as responder cells. Target cells are syngeneic with stimulator cells (H-2 ^b )
Leukemia cells derived from C57BL/b mice, EL4, were used. ¹⁰⁶ EL4 cells with ₁₀₀ ^μCi _Na251CrO4
(1 mCi/ml) was used to incorporate ⁵¹ Cr into the cytoplasm by incubation at 37° C. for 1 hour, followed by washing, adjusting the concentration to 10 ⁴ cells/ml, and using the cells as target cells. Measurement of cytotoxic activity is performed using 0.1ml of responder cell suspension.
Add 0.1 ml of the target cell suspension to a 96-well flat-bottomed plate, and after culturing at 37°C for 4 hours, measure the amount of ⁵¹ Cr released into the supernatant, and calculate the cytotoxic activity using the following formula: did. Cytotoxic activity (%) = [responder cells + target cells, cpm)
- (Target cells alone, cpm) / (Total radioactivity, cpm) - Target cells alone, cpm)] × 100 In addition, the cytotoxic T induced by the above method
The cells showed strong cytotoxic activity against stimulator cells (H- ^2b ) and syngeneic EL4 cells (H- ^2b ), but against allogeneic Meth A cells (H- ^2d ). H-2 ^b showed no cytotoxic activity at all.
It was suggested that these cells were restricted allogeneic cytotoxic T cells. As a result of adding the immunosuppressive substance of the present invention at a concentration of 0.01 to 1 μg/ml and examining the effect on induction of allogeneic cytotoxic T cells, as shown in Table 6, almost no cytotoxic activity was observed. The induction of allogeneic cytotoxic T cells was significantly suppressed by the immunosuppressive substance of the present invention.

[Table] Experimental Example 7 (Suppressive effect on proliferation of IL3-dependent mouse cell line FDCP2 induced by interleukin-3 (IL3)) IL3-dependent mouse cell line FDCP2 was grown in RPMI1640 medium containing 10% fetal bovine serum. The concentration was adjusted to 2×10 ⁵ cells/ml. 100μ of this cell suspension and 50μ of mouse leukemia cell WEHI3 culture supernatant containing IL3 were added to each well of a 96-well microtest plate in which 100μ of the test substance had been previously placed, and the mixture was heated at 37℃ for 5 minutes.
% carbon dioxide gas for 20 hours. After culturing, 0.5 μCi/well of ³ H-thymidine was added, and after further culturing for 4 hours under the same conditions, the cells were collected using a cell harvester and the radioactivity incorporated into the cells was measured using a liquid scintillation counter. This was used as an indicator of IL3-dependent proliferation. As shown in Table 7, the immunosuppressant of the present invention suppresses the increase in ^3H -thymidine uptake in FDCP2 cells induced by IL3 (approximately 60% at a concentration of 1 μg/ml).
%), suggesting that the immunosuppressive substance of the present invention has the activity of suppressing IL3-dependent proliferation.

[Table] Experimental Example 8 (Suppressive effect on interleukin 1 (IL1) response of mouse thymus cells) The thymus was removed from a 7-week-old male C3H/H ₂ N mouse, and single cells were cultured using serum-free RPMI1640 medium. It was made into a floating liquid. After washing three times with the same medium, 20% fetal bovine serum 5 x 10 ^-5 M/2-mercaptoethanol, 2
×10 ^-3 M/L-glutamine, 1 × 10 ^-3 M sodium pyruvate phytohemagglutinin (PHA,
Contains 1 μg/ml (Wellcome, HA16/17) and 2 units/ml of Human Ultrapure Interleukin 1 (GUPi-1, Genzyme)
They were suspended in RPMI1640 medium at a concentration of 1.5 x 10 ^-7 cells/ml. A solution containing 100μ of this cell suspension and the immunosuppressive substance of the present invention or cyclosporin A
100μ of 3H-thymidine was mixed in each well of a 96-well flat-bottomed microtest plate, and cultured for ⁶⁶ hours at 37°C and 5% carbon dioxide.
0.5 μCi/well was added and cultured for an additional 6 hours. After the culture was completed, the cells in each well were collected onto a filter using a multiple cell harvester, and the radioactivity incorporated into the cells was measured by a liquid scintillation method using a toluene-based scintillator. The results obtained by the above method are shown in Table 8. In the table, SD represents standard deviation. In addition, the inhibition rate (%) was calculated using the following formula. Suppression rate (%) = [1-
( ^3H -thymidine uptake when test solution is added to PHA+IL1) - ( ^3H -thymidine uptake of PHA alone)/(PHA+I
^3H -thymidine uptake of L1) - ( ^3H -thymidine uptake of PHA alone) ×100

[Table] Epidemic suppression substances
2 3973±39 91.
8
0.2 4646±826 83.9

Claims (1)

[Scope of Claims] 1. A method for producing an immunosuppressive substance, which comprises culturing an immunosuppressive substance-producing bacterium belonging to the genus Isaria and collecting the immunosuppressive substance from the culture. 2 Claim 1 wherein the immunosuppressive substance is myriocin
Method for producing the immunosuppressive substance described. 3. Claim 1 or 2, characterized in that the immunosuppressive substance-producing bacterium is Isaria cinclairii.
Method for producing the immunosuppressive substance described.