KR920005811B1 - Immunosuppressant - Google Patents

Abstract

No content.

Description

Immunosuppressant

The present invention relates to novel uses of extracts of certain microorganisms and their homologues, including the genus Isaria, and to novel compounds associated with them.

Cyclosporine is mentioned as an immunosuppressant known so far. Cyclosporin has been used for the purpose of suppressing the rejection of kidney transplantation, and has an excellent immunosuppressive effect.

However, cyclosporin has the disadvantage of accompanying side effects (eg renal failure, liver failure).

Thus, there has been a need for immunosuppressants with potent immunosuppressive activity and at the same time with as few side effects as possible.

In view of the above, the inventors have conducted extensive studies and found that the compounds of the formula (I) including the novel compounds and their lactones have fewer side effects while having excellent immunosuppressive activity. The present invention has been completed.

That is, the present invention provides an immunity comprising an effective amount of at least one compound selected from compounds of formula (I) (hereinafter referred to as compound (I)) and lactones thereof, and a pharmaceutically acceptable carrier. Inhibitor; A method for preventing and treating autoimmune diseases or suppressing rejection, characterized in that an effective amount of at least one compound selected from compound (I) and lactones thereof is administered; And compounds of the following formula (I-1) and lactones thereof.

는 단일결합 또는 이중결합을 나타낸다)(Wherein R is a hydrogen atom or acyl, Y is carbonyl or hydroxymethyl,

Represents a single bond or a double bond)

Examples of R as acyl described herein include alkanoyl having 2 to 5 carbon atoms such as acetyl, propionyl, butyryl, pivaloyl and the like, and aromatic acyl having 7 to 11 carbon atoms such as benzoyl, phenylacetyl, etc. .

Preferred compound (I) and lactones thereof are as follows.

Of the above compounds, the last two compounds, namely the compound of formula (I-1) and its lactones, are novel compounds.

이 이중결합이며, Y가 카르보닐인 식(Ⅰ)의 화합물, 즉 ISP -I은 항진균작용을 갖는 마이리오신(Myriocin) 또는 테르모자이모시딘(Thermozymocidin)으로서 공지되어 있다.Obtained according to Example 1 described below, R is a hydrogen atom,

The compound of formula (I), i.e., ISP-I, which is this double bond and Y is carbonyl, is known as Myriocin or Thermozymocidin with antifungal action.

[Reference: The Journal of Antibiotics, Vol. 25, No. 2, 109-115 (1972), The Journal of Organic Chemistry, 38 (7), 1253-1260. (1973), J. Chem. Soc. Perkin Trans. I, 1613-1619 (1983), J. Chem. Soc., Chem. Commun., 488-490 (1982), and the like.

Compound (I) and their lactones can be produced, for example, by a fermentation method or a synthesis method according to Production Method 1-5 described below.

The preparation of ISP-I has already been published in the cited documents mentioned above.

Preparation Method 1 (Fermentation Method)

ISP-I can typically be obtained by fermenting ISP-I-producing microorganisms and harvesting ISP-I from the culture. Examples of the microorganisms to be used include, for example, those belonging to Ascomycotina and Deuteromycontina, and more specifically, to the genus Isaria and Mycelia belonging to Deuteromycotina and Mycelia and Ascomai. Myriococcus (Tyelavia) belonging to Cortina, each of which belongs to Isaria Sinclairii ATCC Accession No. 24400, Myriococcum albomyces ATCC 16425 and Mycelia Sterilia ATCC Accession No. 20349 are deposited with the American Type Culture Collection.

ISP-I is also produced by mutants which can be obtained by modifying the above-mentioned strains using conventional artificial mutants such as ultra-ultraviolet irradiation, microwave irradiation and chemical treatment.

ISP-I can be cultured in a variety of culture media containing nutrient sources for conventional fungi. For example, carbon sources, such as glucose, starch, glycerin, gingko jelly, dextrin, molasses, maltose and xylose, for the purpose of helping microorganism growth and promoting the production of ISP-I; Nitrogen sources such as corn steep liquor, peptone, yeast extract, potato extract, gravy, soybean wheat, wheat germ, potassium nitrate, sodium nitrate, ammonium sulfate, casein, gluten wheat, cottonseed wheat, feather powder as inorganic or organic nitrogen compounds; Other common inorganic salts; And conventional culture additives such as organic and inorganic materials and antifoaming agents.

There is no specific limitation on the culture method itself, but aerobic immersion culture is more advantageous. For strains belonging to the genus Izaria, the preferred incubation temperature is 20-35 ° C., more preferably 25-30 ° C., and for strains belonging to the genus Myriococum or Mycelia, it is 30-50 ° C. and 35-45 ° C. desirable.

ISP-I produced in culture can be harvested from the culture using conventional methods such as extraction, adsorption, or in combination with these conventional methods. For example, in the case of Isaria sinclilyi belonging to the genus Isaria, insoluble substances such as cells are removed by filtration or centrifugation, and the remaining culture filtrate is contacted with Amberlite XAD-2 to adsorb ISP-I. It can collect by making it. The ISP-I obtained was dissolved in methanol and subjected to reverse phase fractionation chromatography on the dissolved ISP-I to obtain a highly purified ISP-I. In the case of strains such as Myriococum albomaises and Mycelia stelia, which belong to the genus Myriococum and Mycelia, the cells are removed from the culture by a method such as filtration and centrifugation, and then moved to the culture filtrate. The same method as for the strains of the genus Lia is carried out. On the other hand, the isolated cells were extracted with methanol, the filtrate was treated with Amberlite XAD-2, and then another purification method such as chromatography or recrystallization gave ISP-I.

Compounds (I) other than ISP-I and their lactones can be prepared, for example, by the following methods.

Preparation Method 2 (Synthesis Method)

The lactones of compound (I) can be prepared by (1) treating the corresponding compound (I), including ISP-I, with an inorganic acid such as hydrochloric acid or an organic acid such as acetic acid, or ② with a tertiary alcohol such as t-amyl alcohol. have.

Reaction ① can be carried out in a solvent (e.g., alcohol such as methanol or ethanol) that is typically inert to the reaction. The reaction temperature is usually in the range 0-50 ° C., preferably around room temperature, and the reaction time is usually 10-30 hours, preferably about 20 hours.

The reaction temperature of reaction ② is usually in the range of 80-150 ° C., preferably in the range of 100-120 ° C., and the reaction time is usually 10-30 hours, preferably about 20 hours.

Preparation Method 3 (Synthesis Method)

이 단일 결합인 화합물(Ⅰ) 및 이들의 락톤은

이 이중결합인 상응하는 화합물(Ⅰ) 또는 그들의 락톤을 수소화시킴으로써 제조할 수 있다.Among compounds (I) including ISP-I

Compound (I) and these lactones which are this single bond

This double bond can be produced by hydrogenating the corresponding compound (I) or their lactones.

The hydrogenation is usually carried out in a solvent (eg, alcohol such as methanol or ethanol) in the presence of a conventional catalyst such as a palladium compound, nickel compound or platinum compound. The reaction temperature is usually in the range of 0-50 ° C., preferably around room temperature, and the reaction time is usually 1-10 hours, preferably about several hours.

Preparation Method 4 (Synthesis Method)

Compound (I) and lactones thereof in which Y is hydroxymethylene can be prepared by activating the corresponding compound (I) or lactones in which Y is carbonyl.

The reduction reaction may be performed using a metal hydrogen complex salt compound such as sodium borohydride or lithium aluminum hydride.

The reaction is usually carried out in a solvent (eg an alcohol such as methanol or ethanol), the reaction temperature is typically 0-50 ° C., preferably around room temperature, and the reaction time is typically 0.5-4 hours, preferably It's about an hour.

Preparation Method 5 (Synthesis Method)

Compound (I) or lactones thereof in which R is acyl can be prepared by isomerizing the corresponding compound (I) or lactones thereof in which R is a hydrogen atom by a known method.

As the acylating agent in the acylation reaction, for example, acid acetic acid, acid halide, active ester and the like can be used.

The acylation reaction can be carried out under known conditions.

Compound (I) and their lactones have good immunosuppressive action, and therefore, anti-reactive inhibitors in organ or bone marrow transplantation, rheumatoid arthritis, systemic lupus erythema, Sjogren syndrome, multiple sclerosis, myasthenia gravis, I-type diabetes, endocrine Prevention or treatment of autoimmune diseases such as eye disease, primary gallbladder cirrhosis, Crohn's disease, glomerulonephritis, sarcoidosis, psoriasis, pemphigus, hypoplastic anemia, spontaneous thrombocytopenic purpura and allergic symptoms, or human body, bovine, horse, dog, mouse Useful as pharmaceutical or medical reagents for rats and the like.

Compound (I) or lactones thereof may be mixed into a carrier, excipient, diluent or the like to form a dosage form such as powders, capsules, tablets, injections and the like that can be administered to patients. It may also be lyophilized into a pharmaceutical composition using known techniques.

The dosage of Compound (I) or their lactones depends on the disease, condition, weight, sex, age, etc., for example, 0.1-10 mg (active) per day is usually used in an adult to suppress rejection during kidney transplantation. ) Can be administered in one to several doses.

EXAMPLE

Hereinafter, the present invention will be described in more detail with reference to examples, provided that the present invention is not limited to these embodiments. Immunosuppressive activity was analyzed by the following methods.

Immunosuppressive activity was analyzed based on various immune responses in mice, rats and human lymphocytes; For example, mouse, rat or human homologous mixed lymphocyte responses (allogeneic MLR) were used to analyze immunosuppressive activity with high sensitivity. The allogeneic MLR refers to the embryonic development of lymphocytes induced by a mixed culture of lymphocytes derived from two individuals that are allogeneic, but differing in major histocompatibility antigens, such as germ cells, lymphoma cells and peripheral blood lymphocytes. This homologous MLR is a response that exhibits a phenomenon that reflects differences in major histocompatibility antigens between donors; For example, embryonic embryogenesis is not observed in lymphocyte mixed cultures taken from identical twins. Thus, homologous MLRs are widely used to select donors and recipients for organ transplantation.

In homologous MLR, lymphocytes usually taken from one of two donors are used as stimulating cells after treatment with X-ray irradiation or mitomycin C for the purpose of inhibiting mitotic proliferation, while donating the other one. Embryonic development of lymphocytes (reactive cells) taken from phosphorus is measured (One Way-MLR).

Immunosuppressive activity can also be measured by measuring activity that inhibits the induction of major histocompatibility antigen-limited cytotoxic T cells in allogeneic MLR.

In addition, the activity of inhibiting embryonic development of lymphocytes induced by stimulation with various mitogens (eg, concanavalin A, plant hemagglutinin, US soybean mitogen, or differentiation of lymphocytes such as T cells and B cells). Immunosuppressive activity by activity of lymphocytes induced by cytokines (e.g., interleukin 1, 2, 3, 4, 5, 6, etc.) or by promoting mitotic proliferation or inhibiting mitotic proliferation Can be evaluated. In addition, the immunosuppressive activity can also be evaluated by the activity of inhibiting the production of the cytokines from T cells, macrophages and the like.

Immunosuppressive activity also inhibits the induction of plasma cells producing anti-heterologous erythroid cell antibodies induced in mouse splenocytes previously immunized by intraperitoneal, oral, intravenous or intradermal injection of double erythrocytes into mice. It can also be assessed by activity, or by inhibiting rejection such as allogeneic mice, transplant-versus-host reactions, delayed allergies, augmented arthritis, etc. in organ transplantation.

Furthermore, the compounds (MRL / lpr mice, NZB / WP ₁ mice or BXSB mice) can be used to produce anti-DNA antibodies, to produce rheumatoid factor nephritis, to inhibit lymphocyte dysplasia, or to develop autoimmune diseases. I) or immunosuppressive activity can also be assessed by the life-extension efficacy observed upon administration of their lactones.

Example 1

(i) Jar culture of Isaiah Sinclilee

The GPY medium of 100ml (1 liters of glucose 30g, peptone 5g, yeast extract _{3g, KH 2 PO 4 0.3g,} K 2 HPO 4 0.3g , and _{MgSO 4 .7H 2 O 0.3g, pH} 5.5) 500ml- long-neck Into each shake flask, autoclave at 121 ° C. for 20 minutes, and incubate the mycelium of about 1 cm 2 of Isaiah Sinclily ATCC Accession No. 24400 grown on potato dextrose agar medium, followed by reciprocating shaking. Incubate at 25 ° C. for 6 days in a vessel (145 rpm, amplitude 8 cm). The resulting cultures were inoculated into 101-fermented jars containing 5 liters of GPY medium described above, followed by aerobic stirring culture (1 VVM, 300 rpm) at 25 ° C. for 10 days.

(ii) Harvesting ISP-I from Isaiah Sinclily Cultures

Cells and insolubles were filtered from 4.5 l of the culture obtained in (i) above to obtain 4.0 l of the culture filtrate. The obtained culture filtrate is passed through an Amberlite XAD-32 column (ømm x 750mm) to adsorb ISP-I. The column is washed with 4.0 l of water. 6 l of methanol is then passed through to elute ISP-I. The eluate is concentrated under reduced pressure, dissolved in 200 ml of ethyl acetate and extracted three times with 200 ml of water each in a separatory funnel.

The extract extracted with water and the extract extracted with ethyl acetate were separately concentrated under reduced pressure and lyophilized to give 2.23 g and 0.34 g of ISP-I, respectively.

(iii) Purification of ISP-I

ISP-I (2.23 g) obtained by extraction with water in (ii) and then lyophilized was dissolved in 5 ml of water and a reversed phase chromatography column (ODS DM-1020T Fuji-Devison Chemicals Company) , Co) (ømm x h 85mm) Elution is started with water and fractional treatment is carried out in a concentration gradient elution step to increase the methanol concentration.

Fractions eluted with 70% methanol were concentrated to dryness under reduced pressure, then dissolved in a small amount of hot methanol and then cooled to give ISP-I crystals. The crystals were again dissolved in hot methanol and recrystallized to give 40 mg of pure ISP-I.

Example 2

(i) Jar culture of Myriococum Albomaises

100 ml of GCY medium (20 g of glucose in 1 liter, 5 g of corn steep liquor, 3 g of yeast extract and 0.5 g of MgSO ₄ .7H ₂ O, pH 6) were placed in each of two 500 ml-length-necked flasks and 20 at 121 ° C. After autoclaving for about a minute, about 1 cm 2 of Myriococum Albomaises ATCC Accession No. 16425 grown on potato dextrose agar medium was incubated, followed by 40 ° C. in a reciprocating shaker (145 rpm, amplitude 8 cm). Incubate for 3 days. The resulting cultures were inoculated in 10 l-fermented jar containing GCY medium and 1 g of anti-foaming agent (F-18 from Dow Coning Co.) and aerobic stirred culture at 40 ° C. for 7 days ( 0.5 VVM, 300 rpm).

(ii) Harvesting ISP-I from Myriococum Albomaises Culture

Cells are filtered from 4.5 l of the culture obtained in (i) above to obtain a filtrate. The culture filtrate (4) is passed through an Amberlite XAD-2 column (ø 40 mm × h 750 mm) to adsorb ISP-I. The column is washed with 1 l of water, and then the eluate containing ISP-I eluted with 1 l of 30% methanol, 1 l of 50% methanol and 3 l of 80% methanol and then eluted with 80% methanol is collected.

Separately, cells were extracted three times with about five times the weight of the cell's wet weight with methanol, water was added to the extract to obtain a 30% methanol solution, and then flowed into an Amberlite XAD-2 column (ø 40 mm × h 750 mm) to ISP- Adsorb I. After flowing 1 l of 30% methanol, 1 l of 50% methanol and 3 l of 80% methanol, the eluate containing Compound (I) eluted with 80% methanol was collected.

Fractions obtained as described above, obtained by elution with 80% methanol from the cells and also from the cells were combined, concentrated under reduced pressure, and lyophilized to obtain 0.5 g of a powder containing ISP-I.

(iii) Purification of ISP-I

The powder containing ISP-I obtained in (ii) (0.5 g) was washed with ethyl acetate and then with hot water (60 DEG C), dissolved in hot methanol and cooled to obtain crystals of ISP-I. . Methanol recrystallization was repeated to give 250 mg of ISP-I.

Example 3

(i) culturing jars of mycelia stelia

Mycelia stelia ATCC Accession No. 20349 is cultured in the same manner as in Example 2 (i).

(ii) Isolation of ISP-I from Mycelia Steliae Culture

In a similar manner as in Example 2 (ii), 1 g of a powder containing ISP-I was obtained.

(iii) Purification of ISP-I

600 mg of ISP-I was obtained by the same method as in Example 2 (iii).

The physical properties of ISP-I obtained in Example 1-3 are as follows.

Example 4

100 mg of ISP-I is dissolved in 20 ml of methanol, 0.4 ml of 44% methanolic hydrochloric acid is added, then left at room temperature overnight, and then the solvent is evaporated under reduced pressure. The residue was purified by silica gel chromatography using a mixture of chloroform and methanol (9: 1) and recrystallized from chloroform-petroleum ether to give 75 mg of the compound having the following structural formula.

Physical properties of the obtained compound are as follows.

Example 5

50 mg of ISP-I is dissolved in 15 ml of methanol, and the solution is subjected to a hydrogenation reaction using 5% palladium carbon (40 mg) as a catalyst. After palladium carbon was filtered off and the solvent was evaporated under reduced pressure, methanol recrystallization was repeated to give 38 mg of the compound having the following structural formula.

Physical properties of the obtained compound are as follows.

Example 6

50 mg of ISP-I is dissolved in 15 ml of methanol and sodium borohydride (20 mg) is added in small portions. After 30 minutes, 1 ml of saturated ammonium chloride solution was added to stop the reaction, and chloroform extraction and methanol recrystallization were carried out to obtain 46 mg of the compound having the following structural formula.

Physical properties of the obtained compound are as follows.

Example 7

The compound obtained in Example 5 was treated in the same manner as in Example 4 to obtain a compound having the following structural formula.

Physical properties of the obtained compound are as follows.

Example 8

The compound obtained in Example 6 was treated in the same manner as in Example 4 to obtain a compound having the following structural formula.

Physical properties of the obtained compound are as follows.

Example 9

The compound obtained in Example 5 was treated in the same manner as in Example 6 to obtain a compound having the following structural formula.

Physical properties of the obtained compound are as follows.

Example 10

The compound obtained in Example 9 was treated in the same manner as in Example 4 to obtain a compound having the following structural formula.

Physical properties of the obtained compound are as follows.

Example 11

100 mg of ISP-I is dissolved in 30 ml of methanol, 10 ml of acetic anhydride is added and then left at room temperature overnight. Water is added to decompose acetic anhydride and the solvent is evaporated under reduced pressure. The residue was purified by silica gel (10 g) chromatography using chloroform-methanol (9: 1) to give 60 mg of the compound having the following structural formula.

Physical properties of the obtained compound are as follows.

Experimental Example

Experimental Example 1: Immunosuppressive Activity of Immunosuppressive Compounds

The mouse allogeneic lymphocyte reaction (hereinafter abbreviated as MLR) was used to assess the immunosuppressive activity of Compound (I) and their lactones. Mouse allogeneic MLR is performed by mixing and culturing BALB / c mouse (H-2 ^d ) splenocytes as reactive cells and mitomycin C-treated C57BL / 6 (H-2 ^b ) splenocytes as stimulating cells in equal amounts.

Reaction cells are prepared as follows.

RPMI 1640 culture medium (60 μg / ml) with 5% addition of heat-inactivated fetal calf serum (hereinafter abbreviated to FCS) after excision of the spleen from 5- to 6-week-old male BALB / c mice Single cell suspensions of splenic cells were prepared using kanamycin sulfate, 2 mM-glutamine, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate (HEPES) and 0.1% sodium bicarbonate). do. After hemolysis, the cell suspension was adjusted to 10 ⁷ cells / ml using 10 ^-4 M 2-metcapto ethynol and RPMI 1640 culture medium supplemented with 20% FCS, and then used as a reaction cell suspension. do.

Stimulator cells are prepared as follows:

After excising spleen from 5- to 6-week-old male C57BL / 6 mice, single cell suspensions of splenocytes are prepared using RPMI 1640 culture medium. After hemolysis, cells are treated with 40 μg / ml mitomycin C at 37 ° C. for 60 minutes. After washing three times, using a RPMI 1640 culture medium supplemented with 10 ^-4 M 2-mercapto ethanol and 20% FCS to adjust the concentration of the cell suspension to 10 ⁷ cells / ml to use as a stimulating cell suspension.

50 µl of the reaction cell suspension obtained as described above, 50 µl of the stimulation cell suspension and 100 µl of the test component are placed in a 96-well microculture plate and incubated at 37 ° C. for 4 days in a 5% carbon dioxide atmosphere.

Analyze embryonic development of lymphocytes using the 3 _H -thymidine uptake method as an indicator. That is, 0.5 μCi / well is added with 3 _H -thymidine after the incubation is completed, and incubated again for 4 hours. Cells are harvested with a cell harvester and the radioactivity incorporated into the cells is measured with a liquid scintillation counter to obtain an embryonic index of lymphocytes in mouse allogeneic MLR. Inhibitory activity of mouse allogeneic MLR is evaluated by calculating the percentage inhibition according to the following equation. The results are summarized in Tables 1A-1D below.

TABLE 1A

TABLE 1B

TABLE 1C

Test components are dissolved in methanol or suspended in a mixture of methanol and acetic acid and diluted with RPMI 1640 culture medium. Methanol and acetic acid were used at concentrations of less than 0.01%, which had no effect on homologous MLR.

Compound (I) and their lactones were examined for the activity of inhibiting lymphocyte embryonic response in mouse allogeneic MLR at a final concentration range of 1 μg / ml-0.001 μg / ml. As shown in Tables 1A-1D above, ISP-I inhibits mouse allergen MLR at 50% -inhibitory concentration (IC ₅₀ ) 7.1 × 10 ⁻³ μg / ml. Other compounds (I) and their lactones also showed comparable activity with ISP-I, and their IC ₅₀ values were about 1/10 or less of cyclosporin A. However, these compounds did not show cytotoxicity against mouse L 929 cells even at a concentration of 10 μg / ml (IC ₅₀ = 10 μg / ml or more).

Lymphogenesis of lymphocytes can also be measured by colorimetric methods using 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) described below.

◎ Colorimetric method using MTT

After the incubation is completed, the supernatant (100 μl) is removed from each well, 20 μl of 5 mg / ml MTT solution is added to each well, and then incubated at 37 ° C. for 4 hours. Then, 100 μl of 0.01 N hydrochloric acid solution containing 10% sodium dodecyl sulfate is added, and the resulting formazan purple crystals are dissolved by incubating at 37 ° C. overnight. The absorbance at 550 mm is measured with a microplate absorption spectrophotometer and used as an indicator of the embryonic response of lymphocytes in mouse homologous MLR. Inhibitory activity of mouse allogeneic MLR is evaluated by calculating the percentage inhibition according to the following formula.

Experimental Example 2: Human homologous MLR-inhibitory activity

Human homologous MLR-inhibitory activity is analyzed as follows:

Peripheral blood lymphocytes obtained by centrifuging Ficoll-Paque density gradients of normal humans were suspended in a RPMI-1640 medium supplemented with 10% FCS on a plastic dish and in a 5% carbon dioxide atmosphere. Incubate at 37 ° C. for 2 hours. After the incubation was completed, gently pipetting to collect and centrifuge the supernatant (1000 rpm, 5 minutes) to obtain cells that do not adhere to the plastic. Cells were not attached to the plastic nylon - after passing the wool column resultant cells that do not adhere to the nylon, the concentration of the cell suspension using the RPMI 1640 culture medium augmented the 10% FCS 4 × 10 ⁶ cells / Adjust to ml.

Cells attached to the plastic are recovered from the plastic dish by phosphate buffered saline supplemented with 5% FCS and 0.02% disodium ethylenediamine-tetraacetic acid (EDTA) followed by strong pipetting. The cells attached to the plastics were treated with 40 μg / ml mitomycin C for 60 minutes at 37 ° C., then washed three times, followed by 4 × 10 ⁶ cells / ml in RPMI 1640 culture medium supplemented with 10% FCS. Suspension is concentrated. The resulting suspension is used as a stimulating cell suspension. 50 µl of the reaction cell suspension obtained from A or C in the air is mixed with 50 µl of the stimulation cell suspension obtained from donor B or D, and 100 µl of the test component is added thereto, followed by 5 at 37 ° C. in a 5% atmosphere of carbon dioxide. Incubate daily.

After the incubation is completed, 1.0 μCi / well of 3 _H -thymidine is added, incubated for 18 hours, and the cells are harvested by a cell harvester. Intracellular radioactivity is measured with a liquid scintillation counter and used as an indicator of the embryonic response of lymphocytes in human homologous MLR. Inhibitory activity of human allogeneic MLR is evaluated by calculating the percentage inhibition according to the following formula.

Compound (I) and their lactones were investigated for their activity in inhibiting the embryonic response of lymphocytes in human homologous MLR in the final concentration range of 10 μg / ml-10 ⁻⁵ μg / ml. As shown in Tables 2A and 2B below, the 50% inhibitory concentration (IC ₅₀ ) in human homologous MLR of ISP-I prepared in Example 1 of the present invention was 1.0 × 10 ⁻⁴ μg / ml, IC ₅₀ of the compound 4 is 7.9 × 10 ^-4 ㎍ / ml, and the IC ₅₀ of the embodiment of the example 6 compound is 2.2 × 10 ^-4 ㎍ / ml, and the IC ₅₀ and the embodiment of the compound of example 5 was 3.5 × 10 ^{- 4} μg / ml.

From the results shown in these Tables 2A and 2B, it can be seen that the IC ₅₀ value in human homologous MLR of Compound (I) and their lactones is lower than that of cyclosporin A.

TABLE 2A

TABLE 2B

Experimental Example 3

Inhibition of Homologous Reactive Cytotoxic T Cells in Mice Allogeneic Lymphocyte Mixed Cultures (MLC)

Spherical cell suspension (2 × 10 ⁷ cells / ml) of BALB / c mice (H-2 ^b ) prepared in the same manner as in Experiment 1 0.5 ml mitomycin C-treated C57BL / 6 mice (H-2 ^d 0.5 ml of splenic cell suspension (2 × 10 ⁷ cells / ml) and 1.0 ml of test ingredient are added to a 24-well multi dish and incubated for 6 days at 37 ° C. in a 5% carbon dioxide atmosphere.

After incubation, cells were harvested by centrifugation, and the concentration of cell suspension was made to 5 × 10 ⁶ -6.25 × 10 ⁵ cells / ml using RPMI 1640 culture medium supplemented with 10% FCS and then operated. Used as cell suspension.

As target cells, leukemia cells EL ₄ obtained from syngeneic (H- ^2b ) C57BL / 6 mice used in the preparation of stimulatory cells are used. 10 ⁶ EL ₄ cells were incubated at 37 ° C. for 1 hour in the presence of 100 uCi of Na ₂ 51 CrO ₄ (ImCi / ml) to inject 51Cr into the cytoplasm. After the cells are washed, they are adjusted to a concentration of 10 ⁴ cells / ml and used as the target cell suspension.

To analyze cytotoxic activity, 0.1 ml of cell suspension and 0.1 ml of target cell suspension are added to a 96-well flat bottom plate and incubated at 37 ° C. for 4 hours. The cytotoxic activity is calculated according to the following formula by measuring the amount of 51Cr released into the supernatant.

Cytotoxic T cells induced by the above-described methods show potent cytotoxic activity against EL ₄ cells (H-2 ^b ) that are synonymous with stimulating cells (H-2 ^b ), while homologous Meth A cells (H-2 _d ) showed no cytotoxicity. Thus, it is inferred that they are H-2 ^b -restricted allo-reactive cytotoxic T cells.

As shown in Table 3 below, the addition of Compound (I) or their lactones significantly inhibited the induction of allogeneic-reactive cytotoxic T cells and almost no cytotoxic activity was observed.

TABLE 3

Experimental Example 4

Inhibition of Embryonic Response of Mouse Splenocytes by Mitogen Stimulation

The effects of mouse splenocytes stimulated with plant coagulation (PHA) or US gelatin soybean mitogen (PWN) on mouse embryonic response were investigated as follows.

After removing the spleen from 5-BA-8 week male BALB / c mice, single cell suspensions of splenocytes are prepared using RPMI 1640 culture medium supplemented with 5% FCS. After hemolysis, the concentration of the suspension was adjusted to 5 × 10 ⁶ cells / ml using RPMI 1640 culture medium supplemented with 10 ⁻⁴ M 2-methcapto ethanol and 20% FCS, where PHA or PWM was added. do. 100 μl of cell suspension is added to each well of a 96-well microculture plate, and then 100 μl of sample solution per unit well (mouse spleen cells 5 × 10 ⁵ per unit well), 37 ° C. in a 5% carbon dioxide atmosphere. After 72 hours of incubation, 0.5 μCi / well of ³ H-thymidine is added and incubated again for 4 hours under the same conditions. After the incubation is completed, cells are collected by a cell harvester, and the radioactivity inherent in the cells is measured by a liquid scintillation counter, which is used as an indicator of embryonic response of mouse splenocytes. The results are summarized in Table 4 below.

TABLE 4

As shown in Table 4 above, Compound (I) and their lactones strongly inhibit the incorporation of ³ H-thymidine induced by PHA or PWM when crossed with control tests without addition of these compounds.

Experimental Example 5

Inhibition of interleukin 1 (IL1) response in mouse thymic cells.

After thymus ablation from 7 week old male C3H / HeN mice, single cell suspensions are prepared using RPMI 1640 culture medium without serum. After washing three times with medium, 20% fetal calf serum, 5 × 10 ⁻⁵ M 2-mercapto ethanol, 2 × 10 ⁻³ M L-glutamine, 1 × 10 ⁻³ M sodium pyruvate, 1 μg / ml RPMI 1640 supplemented with plant agglomerates (PHA, welcome Co., (HA 16/17) and 2 units / ml of human ultrapure interleukin 1 (Genzyme Co., GUPi-1) The cells are suspended in culture medium at a concentration of 1.5 × 10 ⁷ cells / ml 100 μl of the cell suspension and 100 μl of the solution containing ISP-I in each well of a micro-culture plate with a flat bottom of 96-well. Mix, incubate for 66 hours at 37 ° C. in 5% carbon dioxide, then add 0.5 μCi / well of ³ H-thymidine and incubate for another 6 hours After the incubation is complete, present in each well The cells were collected on the filter using a multiple cell counter, and the radiation incorporated into the cells by the liquid scintillation method using a toluene-based scintillator. Measure your ability.

The results obtained are summarized in Table 5 below. In Table 5, SD means standard deviation. % Inhibition is calculated according to the following formula.

TABLE 5

As shown in Table 5, it is evident that Compound (I) and their lactones are dose dependent and inhibit the IL-1 response.

Experimental Example 6

Inhibition of IL2 Production in Mice Allogeneic Lymphocyte Mixed Cultures (MLC) and PHA-stimulated Mouse Splenocytes

Mice homologous MLC was performed as follows: Reaction cell suspension, stimulation cell suspension (0.5 ml each) and test ingredient 1 ml prepared in the same manner as in Experimental Example 1 were added to a 24-well dulti dish, and 5% carbon dioxide atmosphere. Incubate at 37 ° C for 2 days. After incubation, the supernatant is harvested and used as supernatant of mouse allogeneic MLC.

PHA-stimulated mouse splenocytes were cultured as follows: PHA (1/100 dilution) was added to the BALB / c mouse splenocyte suspension prepared in the same manner as in Experimental Example 4. The cell suspension (1 ml) and 1 ml of test ingredient are added to a 24-well multi dish and incubated for 24 hours at 37 ° C. in a 5% carbon dioxide atmosphere. After the incubation is complete, the supernatant is collected and used as the supernatant of the PHA-stimulated culture.

IL2 activity of supernatants of mouse allogeneic MLC and supernatants of PHA-stimulated mouse splenic cell cultures was analyzed as follows: CTLL-2 cells, IL2-associated mouse cell line, were cultured in RPMI 1640 culture medium supplemented with 30% FCS. Suspension at 10 ⁵ cells / ml, 100 μl of the suspension is added to each well of a 96-well microculture plate, and 100 μl of the MLC supernatant described above is injected. After incubation at 37 ° C. for 20 hours in a 5% carbon dioxide atmosphere, 0.5 μCi / well of ³ H-thymidine is added and incubated for 4 hours under the same conditions. After the incubation is over, the cells are harvested with a cell counter and the radioactive aspirated in the cells is measured using a liquid scintillation counter. The incorporation of ³ H-thymidine showed IL2 activity at titers (U / ml) at different concentrations up to 50%. The results are summarized in Table 6 below.

TABLE 6

As shown in Table 6 above, it is demonstrated that Compound (I) and their lactones inhibit IL2 production in mouse allogeneic MLC and PHA-stimulated mouse splenocytes.

Experimental Example 7

Inhibition of the Incorporation of IL-2 Induced ³ H-thymidine in the IL2-Related Mouse Cell Line CTLL-2

The IL2-associated mouse cell line CTLL-2 cells are suspended in RPMI 1640 culture medium supplemented with 30% FCS at a concentration of 2 × 10 ⁵ cells / ml. 50 [mu] l of the concanavalin A-stimulated rat splenocyte culture supernatant containing the suspension (50 [mu] l) and IL2 is added to each well of a 96-well microculture plate, and 100 [mu] l of test component is added thereto. After incubation at 37 ° C. for 20,44 or 68 hours in 5% carbon dioxide atmosphere, 0.5 μCi / well of ³ H-thymidine is added and incubated for 4 hours under the same conditions. After the incubation is completed, cells are collected by a cell counter and the radioactivity incorporated into the cells is measured using a liquid scintillation counter. The results are summarized in Table 7 below.

TABLE 7

As shown in Table 7, compound (I) and their lactones strongly inhibit IL2-induced ³ H-thymidine incorporation of CTLL-2 cells.

Experimental Example 8

Inhibition of Interleukin Direceptor (IL-2R-Tac) Expression in Mouse Allogeneic MLC and PHA-stimulated Mouse Spleen Cells

Mouse allogeneic MLC and PHA-stimulated mouse splenocytes were prepared in the same manner as in Experimental Example 3.

IL-2R (Tac) induced in mouse allogeneic MLC and PHA-stimulated mouse splenocytes was analyzed as follows.

Mice homologous MLC and PHA-stimulated mouse splenocytes were incubated for 24 hours at 37 ° C. in a 5% carbon dioxide atmosphere, followed by centrifugation (1,000 rpm, 5 minutes, 4 ° C.) to harvest the cells and about 10 ⁶ cells. 30 minutes under ice-cooling in 10 μl of phosphate buffered saline (PBS) to which 0.02% sodium azide-containing rat anti-mouse LI-2R monoclonal antibody (40 μg / ml from Boehringer Co.) was added. Incubate. After three washes with ice-cold PBS containing 0.02% sodium azide, 50 μl of fluorescent isothiocyanate (FITC)]-labeled goat anti-rat immunoglobulin G antibody was added and ice-cooled for 30 minutes. Incubate. After washing three times with ice-cooled 0.02% sodium azide-added PBS, 3 drops of PBS2, containing 0.2% sodium azide and 50% glycerin, were added dropwise to the cell pellet. Samples are placed on non-fluorescent slide glass and the IL-2R positive cells are counted under fluorescence microscopy to determine the ratio of IL-2R positive cells to total cell numbers.

TABLE 8

As shown in Table 8 above, Compound (I) and their lactones inhibit the expression of IL-2R induced by stimulation with homologous antigens and PHA in a concentration-involved manner.

Experimental Example 9

Inhibition of IL3 Production in Mice Allogeneic MLC and PHA-stimulated Mouse Splenocytes

Supernatants of mouse allogeneic MLC and supernatants of PHA-stimulated mouse splenocyte cultures were prepared in the same manner as in Experimental Example 6.

The IL3 activity of the supernatants described above was analyzed as follows: IL3-associated mouse cell line FDC-P2 cells were suspended at 10 ⁵ cells / ml in RPMI 1640 culture medium supplemented with 10% FCS and 96-well microculture 100 μl of the suspension is added to each well of the plate, and then 100 μl of the 2-fold serial dilution of the supernatant described above into each well. After incubation for 20 hours at 37 ° C. in 5% carbon dioxide atmosphere, 0.5 μCi / well of ³ H-thymidine is added and incubated again for 4 hours under the same conditions. After the incubation is finished, the cells are collected by a cell harvester, and the radioactivity incorporated into the cells is measured by a liquid scintillation counter. IL3 activity was expressed in titers (U / ml) at concentrations up to 50% incorporation of ³ H-thymidine. The results are summarized in Table 9 below.

TABLE 9

As shown in Table 9 above, Compound (I) and their lactones inhibit IL3 production in mouse allogeneic MLC and PHA stimulated mouse splenocytes.

Experimental Example 10 Inhibition of Proliferation of IL3-Related Mouse Cell Line FDC-P2 Induced by Interleukin 3 (IL3)

The IL3-associated mouse cell line FDC-P2 cells are suspended at a concentration of 2 × 10 ⁵ cells / ml in RPMI 1640 culture medium supplemented with 10% FCS. 100 μl of the suspension and 50 μl of culture supernatant of mouse leukemia cells WEHI3 containing IL3 are added to each well of a 96-well microculture plate containing 100 μl of test components. After incubation at 37 ° C. for 20 hours in a 5% carbon dioxide atmosphere, 0.5 μCi / well of 3 _H -thymidine is added and further incubated for 4 hours under the same conditions. After the incubation is complete, cells are harvested by a cell harvester, and the radioactivity incorporated into the cells is measured with a liquid scintillation counter, which is then used as an indicator of IL3-involved proliferation.

As shown in Table 10 below, Compound (I) and their lactones inhibit the increase of incorporation of IL3-derived 3 _H -thymidine into FDC-P2 cells. Thus, it can be seen that the immunosuppressive compounds according to the invention have the activity of inhibiting IL3-involved proliferation.

TABLE 10

(In the above table, the symbols '+' and '-' mean the presence and absence of IL3, respectively)

Example 11 Inhibition of Interleukin 6 (IL6) Reaction in Mouse Spleen Cells

The spleen was excised from 8-week-old male BALB / c mice to make a single cell suspension in RPMI 1640 culture medium without serum, and then the suspension was centrifuged (1000 rpm, 5 minutes) to obtain cell pellets. . Then, a mixture of 9 parts of 0.16 M ammonium chloride solution and 1 part of 0.17 M Tris solution (pH 7.65) is added to decompose red blood cells, and the resulting suspension is washed three times with RPMI 1640 culture medium containing no serum. The obtained cells were subjected to T24 human bladder sarcoma cell line as 20% FCS, 5 × 10 ⁻⁵ M 2-mercapto ethanol, 2 × 10 ⁻³ M L-glutamine, 1 × 10 ⁻³ sodium pyruvate, and an interleukin 6 member. Suspend the solution at 5 × 10 ⁶ cells / ml in RPMI 1640 culture medium supplemented with% culture supernatant.

100 μl of the cell suspension and 100 μl of the test component are mixed in each well of a 96-well microculture plate and incubated for 72 hours at 37 ° C. in a 5% carbon dioxide atmosphere. After incubation is completed, 50 μl of cell suspension is collected from each well. 100 μl of RPMI 1640 culture medium supplemented with 0.7% agarose gel, 20 μl of saline containing 40% protein A-bound erythrocytes, and 20 μl of 300-fold diluted anti-mouse IgG antiserum with saline After mixing in vitro, spread evenly on a Rohduck plate (Falcon Co. 1034). After holding at room temperature for a few minutes to form a gel, the plates are incubated at 37 ° C. for about 4 hours in a 5% carbon dioxide atmosphere. To each plate, 300 µl of a 40-fold dilution of guinea pig complement in RPMI 1640 culture medium without serum is added and incubated for another 2 hours. The number of plaques formed is then counted under stereomicroscope.

The results of the experiments are summarized in Table 11 below. SD in Table 11 represents the standard deviation, the percent inhibition is calculated according to the following equation.

TABLE 11

From Table 11 above it is demonstrated that Compound (I) and their lactones have the effect of inhibiting IL6 response depending on their dosage.

Experimental Example 12 Inhibition of Mouse Anti-sheep Red Cell Cell Antibody Production

Five to seven-week-old male BALB / c mice are immunized with positive red blood cells (SRBC) and the spleen is excised after three or four days. The number of cells forming plaques (PFCs) was then counted and the efficacy of inhibiting the production of anti-positive erythrocyte cell antibodies was investigated as follows.

[Experiment 1:]

BALB / c mice are immunized with SRBC (1 × 10 ⁷ cells / mouse, intravenous administration) and the ISP-I obtained in Example 1 is administered intraperitoneally for 4 consecutive days from the day of immunization. Four days after the immunization (the day after the final dose), the spleen is excised and the anti-positive erythroid cell-producing cells are measured by the direct plaque method. At the same time the weight of the mice, the wet weight of the thymus and spleen and the number of spleen cells are also measured.

[Experiment 2:]

BALB / c mice are immunized with SRBC (5 × 10 ⁷ cells / mouse, intravenous administration) and the compound obtained in Example 5 is administered intraperitoneally for 4 days continuously from the day of immunization. Four days after the immunization (the day after the final administration), the spleen is excised and the anti-positive erythroid cell-producing cells are measured directly by the plaque method. At the same time the weight of the mice, the wet weight of the thymus and spleen and the number of spleen cells are also measured.

TABLE 12A

TABLE 12B

As shown in Tables 12A and B, Compound (I) and their lactones have the effect of inhibiting antibody production of anti-positive erythrocytes. That is, the number of PFCs per unit splenic cell count (1 × 10 ⁷ cells) and the number of PFCs for the total splenic cell count are reduced.

Experimental Example 13 Inhibition of Allogeneic-Reactive Cytotoxic T Cells Induced by Mice Immunized with Allogeneic Cells

Male BALB / c mice were immunized with C57BL / 6 with mouse splenocytes (5 × 10 ⁷ cells / mouse, intraperitoneally) and 0.1 mg / day from the day following immunization with the ISP-I obtained in Example 1 Six doses are administered intraperitoneally at a dose of kg / day. After 8 days of immunization, spleens were excised to obtain effector cells, and then cytotoxic activity was measured by performing a ⁵¹ Cr release test as in Experiment 3 using EL4 cells as target cells.

As shown in Table 13 below, induction of allogeneic-reactive cytotoxic T cells is inhibited by administering Compound (I) or lactones thereof.

TABLE 13

Experimental Example 14 Cytotoxicity to Mouse L929 Cells

Cytotoxicity to mouse L929 cells was analyzed as follows: L929 cells were suspended at 1.5 × 10 ⁵ cells / concentration in F12 culture medium supplemented with 10% FCS. 100 μl of the suspension is added to each well of a 96-well microculture plate, incubated for 24 hours at 37 ° C. in a 5% carbon dioxide atmosphere, then 100 μl of the test solution is added thereto, followed by further culture for 48 hours. After incubation, 100 µl of the supernatant was taken and the absorbance at 550 nm was measured in the same manner as in the colorimetric method carried out using MTT in Experimental Example 1. The inhibition rate is calculated according to the following formula, which is used as an index indicating cytotoxicity.

The results are summarized in Tables 14A and B below.

TABLE 14A

TABLE 14B

As shown in Tables 14A and B, Compound (I) and their lactones show very low cytotoxicity against mouse L929 cells.

Experimental Example 15 Cytotoxicity to Various Tumor Cell Lines

Cytotoxicity against human tumor cell lines was investigated as follows:

Human cell line cells K562, MOLT4, U937, HL60, KATO III, KB, PC-6, PC-14 and CCRF-CEM, respectively, in RPMI 1640 culture medium supplemented with 20% FCS at 2 × 10 ⁵ cells / concentration Suspend. 50 μl of the suspension is added to each well of a 96-well microculture plate containing 50 μl of test solution. After incubating for 72 hours at 37 ° C. in a 5% carbon dioxide atmosphere, the absorbance at 550 nm was measured in the same manner as in the colorimetric method performed using MTT in Experimental Example 1, and the inhibition rate was calculated in the same manner as in Experimental Example 14. As a cytotoxic indicator. As can be seen from Table 15A-15F below, which shows the calculation results, the cytotoxicity of compound (I) and their lactones to various cultured human tumor cell lines was very weak so that 50% inhibition rate (IC ₅₀ ) was 10. More than μg / ml.

TABLE 15A

TABLE 15B

TABLE 15C

Table 15D

TABLE 15E

Table 15F

[Example]

(1) soft capsules (per capsule)

30 mg of the compound of Example 1

Polyethylene Glycol-300 300mg

Polysorbate 80 20mg

350 mg total

[Method of Preparation]

Polyethylene glycol-300 and polysorbate 80 are added to the compound of Example 1 and the resulting mixture is filled into soft capsules.

(2) Injectables (10 ml per unit ampoule)

0.3% compound of Example 1

Polyethylene Glycol-300 20%

Ethanol 60%

Sufficient amount of distilled water to make 10 ml whole.

[How to prepare]

Ethanol and polyethylene glycol-300 were added to the compound of Example 1 to dissolve, and a sufficient amount of distilled water was added thereto to make a total volume of 10 ml.

As a result, an injection containing 3 ml of the compound of Example 1 in 10 ml of the solution in the ampoule is obtained.

Claims (3)

An immunosuppressive agent comprising an effective amount of a compound having the formula and at least one compound selected from lactones thereof and a pharmaceutically acceptable carrier:

(Wherein R is a hydrogen atom or acyl, Y is carbonyl or hydroxymethylene,

Represents a single bond or a double bond. The immunosuppressive agent of claim 1 comprising an effective amount of at least one compound selected from compounds having the formula:

Compounds having the formula or lactones thereof: