JP2544734B2 - Immunosuppressant - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Industrial Field of the Invention (Wherein R ₁ to R ₉ represent -H, -OH, -OCH ₃ ) or a sugar moiety is a monosaccharide or a disaccharide, and a glycoside of the above flavonoid compound is used as an active ingredient. It relates to immunosuppressants.

PRIOR ART AND PROBLEMS Recently, cyclosporin A has started to be used as an effective immunosuppressant, and the survival rate of transplanted organs has been further improved as compared with the conventional combined use of azathioprine and prednisolone. However, there are many complications associated with the use of cyclosporin A. In particular, the frequent occurrence of renal damage due to the use of cyclosporin A has become a new problem [see, for example, Clinical Immunity, Vol. 14, p. 837 (1982)]. Expectations for organ transplantation, including kidney transplantation, are expected to increase in the future, and it is therefore necessary to develop an effective anti-rejection agent with fewer side effects.

Means for Solving Problems, Actions and Effects The present inventors have found that flavonoid compounds or their glycosides produce antibodies in vitro (in vitro) studies.
The present invention has been completed by discovering that it is an immunosuppressive agent that has an effect of suppressing T cell proliferation and killer T cell differentiation by mixed lymphocyte culture and is applicable to transplant immunity and the like.

The flavonoid compound used in the present invention has the structural formula: (Wherein R ₁ to R ₉ may be the same or different and each represents —H, —OH or —OCH ₃ ).
Examples of these flavonoid compounds include myricetin, morin, kenferrol, huisetin, datiscetin, robinetin, hervacetin, quercetagetin, hibistine, flavone, platolin, chrysin, apigenin, acasetin, luteolin, diosmethine, scutellarein, chrysoeriol and the like. Any sugar component may be used to form these glycosides,
For example, glycosides include glycosides in which the sugar moiety such as glucoside, galactoside, frucside, rhamnoside, arabinoside and xyloside is a monosaccharide, and glycosides in which the sugar moiety such as rhamnoglucoside and rutinoside are disaccharides. Can be mentioned.

These flavonoid compounds and their glycosides have equivalent immunosuppressive activity regardless of whether they are separated and purified from natural plants or chemically synthesized.

The immunosuppressive agent of the present invention is particularly effective in suppressing transplantation immune reaction, and suppresses the function of immunocompetent cells, that is, suppresses production of effector cells. Examples of the inhibitory effect on the rejection reaction from the host to the graft include suppression of cytotoxic T cell generation against alloantigen, suppression of production of effector cells involved in delayed type hypersensitivity, generation of natural killer cells, lymphokine-activated killer cells. Suppression, suppression of lymphokine production and release of interleukin-2 and interleukin-3, suppression of mixed lymphocyte reaction (MLR) and the like. In addition, suppression of the immune reaction from the graft to the host includes, for example, suppression of graft-versus-host immune reaction (GVHR).

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

Example 1 Splenocytes of BXSB / MpJ mice (6 months old, female and male) and splenocytes of BALB / C mice treated with mitomycin C were mixed and cultured with various concentrations of myricetin for 5 days to generate allokiller T cells. The effect of myricetin on the reaction was investigated.

The culture conditions were BXSB splenocytes 5 × 10 ⁶ and BALB / C splenocytes 5 × 10 ^5.
Was added to each well, and 2 ml of 5% fetal bovine serum, 2 mM L-glutamine, and 5 × 10 ⁻⁵ M 2-mercaptoethanol were added to RPMI 1640 culture medium at 37 ° C., 5% carbon dioxide gas-95%. Cultured under air. Myricetin is added at the start of culture and recognizes the produced BALB / C alloantigen (H-2d).
The cell activity was measured by the following method. Has H-2d antigen
P815 mastcytoma cells were labeled with ⁵¹ Cr, 10 ⁴
The cells were mixed with 3 × 10 ⁵ BXSB cells cultured for 5 days and cultured in a 96-well microplate at 37 ° C. for 4 hours, and then ⁵¹ Cr released from P 815 cells destroyed by the allokiller T cells.
The amount was measured and the breaking activity was expressed as a relative value of the amount of ⁵¹ Cr. This calculation formula is as follows.

The results are shown in Table 1. As these results show, at the myricetin concentration of 9.1 × 10 ^-3 to 9.1 × 10 ^-2 μg / ml, an effect of slightly enhancing allokiller cell production was observed.
At μg / ml, a strong inhibition of the allokiller cell formation reaction was observed.

Example 2 In place of myricetin of Example 1, various flavonoid compounds were used, and the effect of the flavonoid compounds on the allokiller T cell generation reaction at these addition concentrations of 9.1 μg / ml was examined.

 The results are shown in Table 2.

Example 3 Instead of myricetin in Example 1, glycosides of various flavonoid compounds were used, and the effect of the flavonoid compounds on the allokiller T cell generation reaction at these addition concentrations of 9.1 μg / ml was examined.

 Table 3 shows the results.

Example 4 Splenocytes of BXMB / MpJ mice (6 months old, female and male) and splenocytes of BALB / C mice treated with mitomycin C 2 × 10 ⁵
Put each into each hole of 96-well microplate, 5% of 0.2 ml
37 in RPMI 1640 culture medium supplemented with fetal bovine serum, 2 ml of L-glutamine, and 5 × 10 ⁻⁵ M 2-mercaptoethanol.
After culturing for 3 days at ℃, 5% carbon dioxide-95% air, 0.
2 μCi (Curie) of ³ H-thymidine was added and cultured for 2 hours, cells were collected by a cell harvester, and the amount of ³ H-thymidine incorporated into cells (decayed per minute, expressed as dpm) was measured, The extent of DNA synthesis was investigated.

Table 4 shows the results. Myricetin concentration is 9.1 x 10
^{At -1} μg / ml, an inhibitory effect on DNA synthesis began to be observed, reaching 9.1 μg
A remarkable inhibitory effect was observed at g / ml.

Example 5 5 × 10 ⁵ spleen cells of BXSB / MpJ mouse (4 months old, female) and 10 ⁵¹ Cr-labeled YAC-1 lymphoma cells (target cells of mouse NK cells) were mixed and mixed at various concentrations. After culturing at 37 ° C for 4 hours in 96-well microplate together with myricetin, by measuring the amount of ⁵¹ Cr released from YAC-1 cells destroyed by NK cells, the effect of myricetin on the target cell destruction reaction activity of NK cells was measured. I investigated the effect. The formula of Example 1 was used as the formula for expressing the strength of NK activity.

The results are shown in Table 5. Myricetin concentration 0.5 μg / ml
From the above, an inhibitory effect on NK activity was confirmed.

Example 6 Instead of myricetin of Example 6, various flavonoid compounds and their glycosides were used, and their addition concentrations were 1.25 μm.
The effect of NK cells on the target cell destruction reaction at g / ml was investigated. The results are shown in Table 6.

Example 7 subjected to mixed lymphocyte culture for 5 days under the same conditions as in Example 1, resulting ^{anti-H-2 d (BALB / C} ) was investigated activity of T _DTH cells. For T _DTH activity, 1 × 10 ⁷ fresh BALB / C splenocytes and 3 × 10 ⁶ cultured cells were mixed, suspended in 30 μl Eagle MEM culture solution, and injected into the foot pads of BALB / C mice. The thickening of the foot pads after the lapse of time was measured with a caliper. The degree of delayed hypersensitivity reaction was evaluated by footpad hypertrophy.

At the myricetin concentration of 10.0 μg / ml, a remarkable inhibition of the T _DTH formation reaction was observed.

Continued Front Page (56) References Biochem. PhavmacoI. 35 [2], (1986), P. 237- 345 Acta Micvobiol. Hung. 32 [4], (1985), p. 377-384 Zh Mikrobiol. Epimiol. Immunobiol. 49 [8], (1972), p. 54-57

Claims (1)

(57) [Claims] 1. A general formula: (In the formula, R ₁ to R ₉ may be the same or different and each represent —H, —OH or —OCH ₃ ), or the flavonoid wherein the sugar moiety is a monosaccharide or a disaccharide. An immunosuppressant comprising a glycoside of a compound as an active ingredient.