JPS61280429A - Immunosuppressant - Google Patents

Abstract

PURPOSE:An immunosuppressant, containing prodigiosin 25-C and/or metacycloprodigiosin as an active constituent and useful for rejection in organ transplantation or treatment of leukemia. CONSTITUTION:An immunosuppressant containing prodigiosin 25-C expressed by formula I and/or metacycloprodigiosin expressed by formula II as an active constituent. A cytotoxic immunosuppressant, e.g. azathioprine, actinomycin C or azaserine, is used for rejection in organ transplantation, but is has serious side effect due to the inhibition of nucleic acid synthesis as a primary site of action. The prodigiosin 25-C and metacycloprodigiosin produced by Streptomyces hiroshimaensis are found to have powerful inhibitory action on lymphoblast formation and inhibitory action on leukocytic cell multiplication derived from T cells and both are useful as immunosuppressants.

Description

Detailed Description of the Invention - No. 1 The present invention provides a method for treating leukemia containing prodediosin 25-C and/or metacycloprodediosin as an active ingredient or for use against rejection reactions to organ transplants. Regarding immunosuppressants.

1. Prodidiocin 25-C and metacycloprodidiocin are used in Streptomyces (t')
It is a red pigment substance produced by red bacterial cells of the 28-24 strain, and is known for its purification method, physical and chemical properties, chemical structure, and antibacterial activity against certain types of bacteria.

(Agricultural and Biological Chemistry (Agr, Biol, Chem,) JVOl
, 31°No, 4. p. 481-489.1987 and Antibiotics Supplement II (1966-1970)
J, 1973, University of Tokyo Press, 1305-1310
page).

However, these documents do not reveal anything about the immunosuppressive effects of prodediocin 25-C and metacycloprodediocin.

Currently, cytotoxic immunosuppressants such as azathioprine, actinomycin C, and azaserine are used to treat organ transplant rejection, but their primary point of action is inhibition of nucleic acid synthesis. Therefore, 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 effect on nucleic acid synthesis and suppresses cell-mediated immunity involving T cells with a certain degree of selectivity.

Based on the above-mentioned grounds, the present inventors used mouse spleen cells to produce concanavalin A (Concanavalin A).
, 7 Phytohemagglutinin (Phytohemag
glutinfn) and ribopolysaccharide (LPS) as mitogens, we have conducted intensive research with the aim of isolating a specific blast suppressant produced by microorganisms, and as a result,
A strong and selective suppressive effect on the concanavalin A response of lymphocytes to the red pigment prodigiosin 25-C and metacycloprodegiosin produced by Streptomyces hiroshimaensis °1LLL, a strain of actinomycetes, derived from Zara T cells. They discovered an inhibitory effect on leukemia cell proliferation and completed the present invention.

. Prodediosin 25-C and metacycloprodediosin, which are the active ingredients in the immunosuppressant of the present invention, are described in “Antibiotics/Supplement n (19E!6-1970)”, 1973, University of Tokyo Press, 1305. It can be obtained by the culture method and purification method described on pages 1-1310.

The inventor's mule, Streptomyces hiroshimaensis (IAM: 0071), was mixed with 2% lactose and 2% glucose.

Corn Stevely force - 3%, sodium nitrate 0.1%.

1-potassium phosphate 0.06%, potassium chloride 0105%
, magnesium sulfate 0.02%, calcium carbonate 0°5
% (separately sterilized), pH 8.5 to 7.0 medium was inoculated, and 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 culture is completed, filter the culture solution, add acetone to the separated wet bacterial cells, stir well, and adjust the pH by adding hydrochloric acid.
Adjusted 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 a substance exhibiting the following physical and chemical properties, which was confirmed to be prodediocin 25-C and metacycloprodegiocin.

(a) Prodediocin 25-C 1) The structural formula of hydrochloride is 7 in dimorphism.
6-78 °C and 105.5-106 °C, free base is 91-91.5 °C0 3) Absorption spectrum λmax = 525 nm (0,1NHC1-MeOH) λ
max=480nm(0,INNaOH-MeOH)(
b) Metacycloprodediosin 1) Structural formula % Formula % 3) Absorption spectrum λmax = 487.530 nm (0.5% KOH-Me
OH) νmax:”IE329, 1801.1507.
1542゜1528s 9 B 1 c m゛1 Prodigiosin 2, which is the active ingredient of the immunosuppressant of the present invention
Although 5-C or metacycloprodediocin may be used alone, it is usually mixed with common excipients or other adjuvants to formulate a dosage form suitable for parenteral administration and preferably oral administration. It is desirable to do so. Preferred dosage forms include, for example, powder, granules, sugar-coated tablets, gun powder, and capsules.

For example, half of the amount can be given. These preparations can be manufactured by conventional methods using, for example, the following excipients or auxiliaries. Lactose, sucrose, various starches, grape 'IN
+ Cellulose, methylcellulose, carboxymethylcellulose, hydroxypropylcellulose.

Magnesium stearate, lauryl sulfate, talc.

Vegetable oils, various polysorbate polyethylene glycols, and mixtures of two or more of these are used. Preparations for oral administration contain 10 to 55% by weight of the active ingredient, and half contain 1 to 55% by weight.
It is desirable to have 3 pups in an amount of 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 1.0 mg/day.
kg is preferred.

1. The immunosuppressive agent of the present invention has an effect of suppressing lymphocyte blastogenesis 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.

Mouse spleen, concanavalin A response suppression effect Mouse lung cells were suspended in RPMI-1840 medium supplemented with tritium-labeled thymidine, 2% calf serum, and 0 to 5 μg/ml concanavalin A. Prodigiosin 25-
C or metacycloprodedioscin 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.

Prodigiosin 25- at each concanavalin A concentration
C. Incorporation of labeled thymidine in the presence of metacycloprodedioscin or cyclosporin A as a comparative drug is shown on a logarithmic scale in FIGS. 1-3, respectively. The vertical axis shows the radioactivity count on a logarithmic scale, and the horizontal axis shows the concentration of each drug on a logarithmic scale.

As is clear from FIG. 3, the effect of cyclosporin A on suppressing the response of concavalin A becomes stronger as the degree of concavalin A is lower. That is, no significant inhibitory effect was observed for either drug at a concentration of 2 μg/ml, which maximizes the mitogen response of lymphocytes. The effects of cyclosporin A are strongly expressed only in the region well below the degree of concanavalin A that gives the maximal mitogenic 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 induced by low concentrations of concanavalin A are weak, and the suppressive effect is stronger at 1 degree concanavalin A (2 μg/ml), which shows the maximum mitogen response. The inhibitory properties of prodigiocin 25=C and metacycloprodigiocin are therefore clearly different from the comparative drug cyclosporin A.

Effect on leukemia cells Using a tumor cell line derived from leukemia, the effect on growth was examined. During the 3-day culture using prodigiosin 25-C or the protein synthesis inhibitor cycloheximide as a comparative drug, live cells and dead cells were distinguished by trypan blue staining every 24 hours. Each drug 1 degree 0
The cell survival rate after 2 days of culture is plotted on the horizontal axis and 5 μg/m I is plotted on the vertical axis, as shown in FIGS. 4 to 7. Figures 4 and 5 are T
Cell-derived leukemia cell lines A/J and L-5178Y
Figure 6.7 shows the cell viability of other leukemia cell lines YAC-1 and P2S5, respectively. As is clear from the figure, prodigiosin 25-C is 0.
At 0.08 μg/ml, it strongly inhibits growth and acts to kill cells.

Disaster] L Kai J Engineering (acute toxicity) When prodigiosin 25-C or metacycloprodigiosin was intraperitoneally administered to ddY male mice (5 weeks old, 9 weight, 26-30 g), the LDEo value was 20 m for both.
g/kg or more.

Mi "L-den", (Formulation example) Formulation example 1 (Granule) Prodediosin 25-C50 (g) Lactose 4000 (g) Hydroxypropylcellulose 500 (g) Starch
500 (g) The above ingredients are mixed and made into granules by a conventional method.

Formulation Example 2 (Capsule) Metacycloprodediocin 10100 (starch x OOO (g) Magnesium stearate 50 (g) The above ingredients were mixed and the active ingredient was placed in a regular gelatin capsule with 10 m of the active ingredient.
Fill 10,000 capsules to contain 10,000 g.

Formulation Example 3 (Tablet) Prodigiosin 25-C200 (g) Lactose
2500 (g) starch
1200 (g) Ethylcellulose 5
0 (1 (g) Magnenium stearate
200 (g) Talc 1
0100 (mix the above ingredients and tablet by a conventional method,
- 20,000 tablets containing 10 mg of active ingredient in the tablet.

m circle 1 Prodediosin 25-C and metacycloprodediosin, which exhibit the above-mentioned effects, can be expected to have therapeutic effects when used orally or parenterally as immunosuppressants and therapeutic agents during organ transplantation.

[Brief explanation of the drawings] Figure 1 shows concanavalin A of prodigiocin 25-C.
(ConA) Shows the characteristics of response suppressing action. In the figure, mu indicates no addition of ConA, and ■ indicates ConAa degree 0.3u.
g/ml l, * is 111 g/ml l, mouth is 2μ
g/ml and O represents 5 μg/ml, respectively. FIG. 2 shows the properties of the ConA response suppressing effect of metacycloprodidioscin. Each mark in the figure indicates the same thing as in FIG. 1. FIG. 3 shows the characteristics of the ConA response suppressing effect of cyclosporin A. Each mark in the figure indicates the same thing as in FIG. 1. FIG. 4 shows the effects of prodigiosin 25-C (marked O) and cycloheximide (marked 0) on T cell-derived cell line A/J. FIG. 5 shows the effects of prodigiosin 25-C (marked O) and cycloheximide (marked 0) on the T cell-derived cell line L5178Y. Figure 6 shows prodigiosin 25-C (○ mark) and cycloheximide (0 mark) against leukemia cell line YAC-1.
This shows the effect of Figure 7 shows prodigiosin 2 against leukemia cell P388.
The effects of 5-C (marked O) and cycloheximide (marked 0) are shown.

Claims (1)

[Scope of Claims] 1) An immunosuppressant containing prodediosin 25-C and/or metacycloprodediosin 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.