JPH0586374B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to an immunosuppressive agent containing prodediosin 25-C and/or metacycloprodediosin as an active ingredient for use in the treatment of leukemia or for use against rejection reactions to organ transplants. PRIOR ART Prodediosin 25-C and metacycloprodediosin are red pigment substances produced by the red cells of Streptomyces strain 28-24, and methods for their purification;
It is known for its physical and chemical properties, chemical structure, and antibacterial activity against certain types of bacteria. (“Agricultural and Biological Chemistry (Agr.Biol.Chem.)” Vol.31, No.4,
p.481-489, 1967 and "Antibiotics Supplement (1966-1970)", published by the University of Tokyo Press, 1973
(pp. 1305-1310). However, from these literatures, prodigiosin25
-C and metacycloprodediocin cannot be found to have any immunosuppressive effects. Problems to be Solved by the Invention Currently, cytotoxic immunosuppressants such as azathioprine, actinomycin C, and azaserine are used to treat organ transplant rejection, but these drugs primarily inhibit nucleic acid synthesis. Because it is the point of action, it has serious side effects. Recently, cyclosporine A has been highly evaluated as a drug that suppresses rejection reactions in organ transplants. The biological action of cyclosporin A is characterized in that it has no significant effect on nucleic acid synthesis and suppresses cell-mediated immunity involving T cells with a certain degree of selectivity. Based on the above basis, the present inventors used mouse spleen cells to develop concanavalin A.
(Concanavalin A), phytohemagglutinin, and lipopolysaccharide (LPS) as mitogenes, we have conducted intensive research with the aim of isolating a specific blast suppression substance produced by microorganisms, and as a result, we have found that The red pigment prodigiocin 25-C and metacycloprodigiocin produced by a strain of Streptomyces hiroshimaensis have a strong and selective suppressive effect on the concanavalin A response of lymphocytes, as well as T cell-derived leukemia. They discovered a cell proliferation inhibitory effect and completed the present invention. Means for Solving the Problems Prodediosin 25-C and metacycle prodegiosin, which are the active ingredients in the immunosuppressant of the present invention, are described in "Antibiotics Supplement (1966-1970)", 1973.
It can be obtained by the culture method and purification method described in 2010, published by the University of Tokyo Press, pp. 1305-1310. The present inventors prepared Streptomyces hiroshimaensis (IAM:0071) with 2% lactose, 2% glucose, 3% corn stew brewer, and sodium nitrate.
0.1%, 1-potassium phosphate 0.06%, potassium chloride
0.05%, magnesium sulfate 0.02%, calcium carbonate 0.5% (separately sterilized), inoculated into a medium with a pH of 6.5 to 7.0,
Shaking culture was performed for 3 days on a reciprocating shaking incubator at a temperature of 26.5°C. This culture solution was transplanted into the above-mentioned medium at a rate of 5%, and cultured with shaking for an additional 7 days. Since the target organism, prodigiosin, is insoluble in water, it is present in the filtration residue containing bacterial cells. After the cultivation was completed, the culture solution was filtered, acetone was added to the separated wet bacterial cells, and after stirring well, hydrochloric acid was added to adjust the pH to 2.0. The produced prodigiosin (red pigment) is extracted into acidic aqueous acetone. The extract obtained by filtering the bacterial cells was concentrated under reduced pressure to remove acetone, and then ethyl acetate was added to the residue for extraction. The extract was dried by adding anhydrous sodium sulfate and then concentrated under reduced pressure to obtain a red oily residue containing prodigiosin. This red residue was dissolved in a small amount of chloroform and subjected to silica gel column chromatography to obtain substances having the following physical and chemical properties, which were confirmed to be prodigiocin 25-C and metacycle prodigiocin. (a) Prodigiosin 25-C 1 structural formula

[C] 2 Melting point Hydrochloride has dimorphism
At 76-78℃ and 105.5-106℃, the free base is
91-91.5℃. 3 Absorption spectrum λmax=525nm (0.1NHCl−MeOH) λmax=460nm (0.1NNaOH−MeOH) (b) Metacycloprodedioscin 1 Structural formula

[Chemical] 2 Melting point Hydrochloride 208-209℃. 3 Absorption spectrum λmax = 467, 530 nm (0.5% KOH-MeOH) νmax = 1629, 1601, 1507, 1542, 1528, 961 cm ^-1 Prodidiocin 25-C or metacycloprodidio which is the active ingredient of the immunosuppressant of the present invention Although the compound may be used alone, it is usually desirable to mix it with common excipients or other adjuvants and formulate it into a dosage form suitable for parenteral and preferably oral administration. Preferred dosage forms include, for example, powders, granules, tablets, sugar-coated tablets, pills, capsules,
Examples include suppositories. These preparations can be manufactured by conventional methods using, for example, the following excipients or auxiliaries. Lactose, sucrose, various starches, glucose, cellulose, methylcellulose,
Carboxymethyl cellulose, hydroxypropyl cellulose, magnesium stearate, lauryl sulfate, talc, vegetable oil, various polysorbate polyethylene glycols, and these two
Mixtures of more than one species are used. Preparations for oral administration preferably contain the active ingredient in an amount of 10 to 55% by weight, and suppositories in an amount of 1 to 50% by weight. When the immunosuppressive agent of the present invention is administered to humans by intravenous injection, the amount of the active ingredient is 0.1 to 0.1 per day.
1.0 mg/Kg is preferred. Effects The immunosuppressant of the present invention has an effect of suppressing lymphoblastogenesis and an effect of suppressing the proliferation of T cell-derived leukemia cells. Examples are shown below, but the present invention is not limited thereto. Example 1 Mouse spleen, concanavalin A response suppression effect Tritium-labeled thymidine, 2% calf serum, and 0 to 5 μg/ml concanavalin A were added.
After suspending mouse spleen cells in RPMI-1640 medium, prodigiosin 25-C or metacycloprodegiosin was added and cultured for 3 hours. After completion, the culture solution was filtered and the radioactivity of thymidine incorporated into the obtained cells was measured. The uptake of labeled thymidine in the presence of prodidiocin 25-C, metacycloprodediocin, or cyclosporin A as a comparative drug at each concanavalin A concentration is shown on a logarithmic scale in FIGS. 1 to 3, respectively. The vertical axis is the radioactivity count shown on a logarithmic scale,
The horizontal axis shows the concentration of each drug on a logarithmic scale. As is clear from Figure 3, Cyclosporin A
The response suppressing effect of concavalin A becomes stronger as the concentration of concavalin A becomes lower. In other words, no significant inhibitory effect was observed for either drug at the concentration of 2 μg/ml, which maximizes the mitogen response of lymphocytes. The effects of cyclosporin A are only strong in the region well below the concanavalin A concentration that gives the maximal mitodiene response. On the other hand, the action characteristics of prodidiocin 25-C and metacycloprodidiocin are shown in Figures 1 and 2. Contrary to cyclosporin A, these suppressive effects on the mitogen response caused by low concentrations of concanavalin A are weak, and the suppressive effects are stronger at the concanavalin A concentration (2 μg/ml), which shows the maximum mitogen response. The inhibitory properties of prodidiocin 25-C and metacycloprodidiocin are therefore clearly different from the comparative drug cyclosporin A. Example 2 Effect on leukemia cells The effect on growth was examined using a tumor cell subculture derived from leukemia. Prodigiosin 25-C
Alternatively, cycloheximide, a protein synthesis inhibitor, was used as a comparative drug, and live cells and dead cells were distinguished from each other by trypan blue staining every 24 hours during the 3-day culture process. The cell viability after 2 days of culture is plotted on the horizontal axis at each drug concentration of 0 to 5 μg/ml, and the 4th to 7th rows are plotted on the vertical axis.
As shown in the figure. Figures 4 and 5 show the cell viability of T cell-derived leukemia cell lines A/J and L-5178Y, respectively, and Figures 6 and 7 show the cell viability of other leukemia cell lines YAC-1 and P388, respectively. Shows survival rate.
As is clear from the figure, prodigiosin 25-C is
At 0.008 μg/ml, it strongly inhibits growth and acts to kill cells. Example 3 (Acute toxicity) When prodigiosin 25-C or metacycloprodigiosin was intraperitoneally administered to ddY male mice (5 weeks old, weight 26-30 g), both _LD50 values were
It was more than 20mg/Kg. Example 4 (Formulation Example) Formulation Example 1 (Granules) Prodedioscin 25-C 50 (g) Lactose 4000 (g) Hydroxypropyl cellulose 500 (g) Starch 500 (g) The above ingredients were mixed and granulated by a conventional method. as a drug. Formulation example 2 (capsules) Metacycloprodedioscin 100 (g) Starch 1000 (g) Magnesium stearate 50 (g) The above ingredients were mixed and packed in a regular gelatin capsule containing 10 mg of the active ingredient. fill it up
10000 capsules. Formulation example 3 (tablet) Prodigiosin 25-C 200(g) Lactose 2500(g) Starch 1200(g) Ethylcellulose 500(g) Magnesium stearate 200(g) Talc 100(g) Mix the above ingredients and prepare by standard method Compress into 20,000 tablets so that each tablet contains 10 mg of the active ingredient. Effects of the invention Prodigiosin 25-C exhibiting the above-mentioned effects
and metacycloprodediocin can be expected to have therapeutic effects when used orally or parenterally as an immunosuppressant and a leukemia therapeutic agent during organ transplantation.

[Brief explanation of the drawing]

FIG. 1 shows the characteristics of the concanavalin A (ConA) response suppressing effect of prodigiosin 25-C. In the figure, ▲ indicates no addition of ConA, and ■ indicates ConA concentration 0.3 μg/ml.
,● means 1μg/ml, □ means 2μg/ml, ○ means 5μg/ml.
ml is shown respectively. FIG. 2 shows the properties of the ConA response suppressive action of metacycloprodidioscin. Each mark in the figure indicates the same thing as in FIG. 1. FIG. 3 shows the properties of cyclosporin A's inhibitory effect on ConA response. Each mark in the figure indicates the same thing as in FIG. 1. FIG. 4 shows the effects of prodigiosin 25-C (○ mark) and cycloheximide (□ mark) on T cell-derived cell line A/J.
FIG. 5 shows the effects of prodigiosin 25-C (○) and cycloheximide (□) on the T cell-derived cell line L5178Y. Figure 6 shows prodigiocin 25-C (○) against leukemia cell line YAC-1.
(marked) and cycloheximide (marked □) are shown. FIG. 7 shows the effects of prodigiocin 25-C (○ mark) and cycloheximide (□ mark) on leukemia cell P338.

Claims (1)

[Scope of Claims] 1. An immunosuppressant containing prodidiocin 25-C and/or metacycloprodidiocin as an active ingredient. 2. The immunosuppressive agent according to claim 1 for use against rejection reactions to organ transplants. 3. The immunosuppressive agent according to claim 1 for use in the treatment of leukemia.