USE OF SPERGUALIN OR A PHARMACEUTICALLY ACCEPTABLE SALT THEREOF FOR THE PREPARATION OF A TRANSPLANTATION IMMUNITY SUPPRESSING AND ANTI-ALLERGIC AGENT

Abstract: The present invention relates to the use of Spergualin or a pharmaceutically acceptable salt thereof for the prepara­ tion of medicaments having transplantation immunity sup­ pressing and anti-allergic activities.

Description: The present invention relates to a transplantation immunity suppressing agent and an anti-allergic agent that contains Spergualin as the effective ingredient and which is suitable for use with organ transplantation. Various immunosuppressants are currently used in order to suppress rejections and other transplantation immunity reactions that may occur in organ transplantation, and among such suppressive agents are steroid hormones, anti-metabolites, alkylating agents and antibiotics. Steroid hormones are effective against a wide variety of allergic diseases, whereas non-steroidal anti-inflammatory agents and anti-inflammatory enzymes are commonly used in symptomatic therapy of inflammations that accompany allergic reactions. Gold compounds and chloroquines are used expressly against rheumatoid arthritis. For the treatment of bronchial asthma and uricaria, anti-histamines are used. Spergualin is a compound that was isolated by Umezawa et al. from the filtrate of a culture broth of Spergualin-producing microorganism of the genus Bacillus (see Japanese Patent Public Disclosure No. 48957/1982). Existing immunosuppressants generally cause serious side effects when they are administered to humans. It is therefore desired to develop immunosuppressive drugs that act selectively on lymphocytes and other cells of immuno-logical importance while causing minimum side effects. On the other hand, in order to cope with the increasing incidence of allergic diseases such as allergic astma, allergic rhinitis and pollinosis, the development of effective anti-allergic agents is desired. The present invention relates to a transplantation immunity suppressing agent and an anti-allergic agent that contains Spergualin or a pharmaceutically acceptable salt thereof (the two are hereunder collectively referred to as "the compound of the present invention") as effective ingredient and which is capable of inhibiting rejections and other transplantation immune reactions that may occur in transplantation of organs and other tissues. Spergualin has the structural formula (I) and may be prepared by a known method (see Japanese Patent Public Disclosure No. 48957/1982): The compound of formula (I) forms salts with acids and from a stability viewpoint, the compound is actually used in the form of its salts. Salt-forming acids may be inorganic or organic if they are pharmaceutically acceptable. Preferred inorganic salts are hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid; preferred organic acids include acetic acid, propionic acid, succinic acid, fumaric acid, maleic acid, malic acid, tartaric acid, glutaric acid, citric acid, benezenesulfonic acid, toluenesulfonic acid, methanesulfonic acid, ethanesulfone acid, propanesulfonic acid, aspartic acid, glutamic acid and sulfamic acid. The compound of the present invention may be directly used as an immunossuppressant or anti-allergic agent, but it is usually administered in admixture with excipients or carriers. Pharmaceutically accetable excipients and carriers should be selected and their type and composition are determined by the route and method of administration. Liquid carriers suitable for preparing injections include water, alcohols, as well as animal, vegetable and synthetic oils such as soybean oil, peanut oil, sesame oil and mineral oil. The following liquid carriers are generally preferred: physiological saline, buffer solutions, sugars such as glucose, mannose arid mannitol, aqueous solutions containing cyclitols such as sugar alcohol and inositol, and glycols such as ethylene glycol, propylene glycol and polyethylene glycol. Alternatively, the compound of the present invention may be freeze-dried together with excipients such as sugars (e.g. glucose, maltose, sucrose, mannose and mannitol), cyclitols (e.g. sugar alcohol and inositol) and aminoacids (e.g. phenylalanine). Before intravenous injection, such freeze-dried compound is dissolved in suitable solvens such as sterilized water, physiological saline, glucose solution, electrolyte solution and amino acid solution. With injections, the content of the effective ingredient generally ranges from 0.1 to 30 wt%, preferably from 1 to 10 wt%. For oral administration, the compound of the present invention may be used in the form of tablets, capsules, powders, granules, liquids or dry syrups. With capsules, tablets and powders, the content of the effective ingredient ranges generally from 5 to 100 wt%, preferably from 25 to 100 wt%. The dosage of the drug containing the compound of the present invention as the effective ingredient should be properly determined depending upon the age and body weight of the patient, as well as the severity and type of the disease. The effective dose generally ranges from 1 to 100 mg/kg·day for parenteral administration, and from 5 to 500 mg/kg·day for oral administration. Acute toxicity test in mice showed that the LD₅₀ of the compound of the present invention was approximately 150 mg/kg for intraperitoneal administration and that the gradual intravenous administration of 80 mg/kg was not lethal. No cumulative toxicity was observed. With these data taken together, the compound of the present invention is considered to offer a highly safe drug (Takeuchi et al., Journal of Antibiotics, vol. 34, No. 12, pp. 1619 - 1621, 1981). The present inventors conducted tests to evaluate the actions of the compound of the invention against the reaction of lymphocyte blast genesis in accordance with the method of Waithe et al. (Waithe et al., Handbook of Experimental Immunology, page 26.1, 1978). The tests revealed that the compound of the present invention inhibited appreciably the T-lymphocyte blast genesis stimulated by concanavalin A (ConA) and the reaction of B-lymphocyte blast genesis stimulated by lipopolysacccharide (LPS) (Tests 1 and 2). Additionally, in accordance with the method of Jerne et al. (Jerne et al., Cell-bound Antibodies, pp. 109 - 122, 1963), the inhibitory action of the compound of the present invention on humoral immunity which is primarily the domain of the B-lymphocytes was examined. The test procedure of counting the number of spleen cells that where extracted form mice sensitized by antigenic sheep red blood cells and which formed immunoglobulin M (IgM) plaques. As summarized in Test 3, the compound of the present invention exhibited strong inhibitory actions. Encouraged by these data which suggest the possiblity that the compound of the present invention would inhibit general antibody production by the B-lymphocytes, the present inventors proceeded with their studies to examine the inhibition of the B-lympocyte ability to produce IgG and IgE antibodies. As summarized in Tests 4 and 5, the compound of the present invention inhibited the production of IgG and IgE. The above results revealed that the compound of the present invention has the ability to inhibit the function of B-lymphoccytes, hence humoral immunity. Therefore, the compound of the invention can be used as the effective ingredient of a transplantation immunosuppressant and an anti-allergic agent for inhibiting rejections and other transplantation immunological reactions that may occur in organ transplantation. In order to demonstrate the possibility of using the compound of the present invention in a transplantation immunosuppressant, such compound was administered to mice transplanted with P815 mastrocytoma as an allograft and checked for any inhibition of the appearance of allokiller T-lymphocytes in response to the graft. As shown in Test 6, when the compound of the present invention was administered for nine consecutive days starting on the day after the transplantation, the activity of the allokiller in the spleen cells was significantly inhibited. In the next place, a skin graft test with rats was conducted. In accordance with the method of Denham et al. (Denham et al., International Archives of Allergy and Applied Immunology, vol. 62, pp. 443 - 458, 1980), the skin of the tail of SHR rats was peeled and allograted onto Fischer rats. When the compound of the present invention was administered intraperitoneally to recipient rats daily, starting on the day after the transplantation, the graft acceptance period for the treated rats was extended significantly in comparison with the untreated group and hence, the compound was found to have an unexpectedly great ability to inhibit rejections due to transplantation immunity (see Test 7). No such side effects as bone marrow inhibition were observed. These results show only one example of the utility of the compound of the present invention in organ transplantation, which is by no means limited to skin transplantation. The inhibitory action of the compound of the present invention on tissue rejections is expected to manifest itself not only in skin transplantation but also in the transplantation of such organs as the kidney, heart, splean, lung, liver, bone marrow secreting organs. The degree of rejection will vary depending upon the type of transplantation, isogenic, allogenic or heterogenic; however, the compound of the present invention is expected to exhibit its effects in all of these cases and differ only with respect to potency. On the basis of the data obtained in Tests 3, 4 and 5 that show the ability of the compound of the present invention to inhibit antibody production, it can be concluded that such compound will be useful in the treatment of many allergic diseases, especially those associated with humoral immunity. The direct causes of allergic diseases differ with the specific type of allergy, but except for delayed type hypertensitivity mediated by sensitized lymphoid cells, all allergic reactions depend on antibody production in response to exogenous and endogenous antigens, and on the resulting antigen/antibody interaction. For example, atopic dermatitis, pollinosis, bronchial asthma and allergicc rhinitis are caused by the immediate hypersensitivity which is associated with humoral immunity, and IgE is believed to play some role in this immediate form of hypersensitivity. As shown in Test 5, when the compound of the present invention was administered daily to mice sensitized by egg albumin, the formation of anti-egg albumin IgE antibodies in the blood remained at significantly low levels throughout the treatment. It was also found that the inhibitory action of the compound lasted for a certain time after the administration of the compound ceased. These data show that the compound of the present invention has a strong inhibitory action on IgE-associated type I allergic reaction and that therefore the compound will offer an anti-allergic drug useful in the treatment of those allergic diseases in which IgE is dissolved. As Tests 4 and 5 show, the compound of the present invention also have the ability to inhibit the production of IgM and IgG, and therefore, its utility will be expanded to the treatment of allergic diseases caused by type II or III hypersensive reaction, such as serum diseases and hemolytic diseases of new born due to Rh incompatibility. The following Test Examples are provided by way of further illustrations of the activities of the compound of the present invention. Test 1: Inhibition of ConA induced reaction of T-lymphocyte blast genesis by the compound of the present invention (trihydrochloride) Spleen cells form BALB/c mice were distributed among wells in a microplate so that each well contained 2 x 10⁵ cells/0.2 ml. The compound was added to all wells but one, the latter being used as a control. ConA (4 µg/ml) was added to all the wells and the so prepared cell suspensions were cultivated in a 5 % CO₂ incubator for 72 hours at 37°C. Eight hours before the completion of the incubation, 1 uCi of ³H-thymidine was added to each and the uptake of the thymidine by the cultured cells was measured with a liquid scintillation counter to estimate the progress of the reaction of T-lymphocyte blast genesis. The percentage inhibition of that genessis by the test compound was calculated by: (1 - Bdpm/Adpm) x 100 wherein Adpm indicates the uptake count for the addition of only ConA and Bdpm, the count for the addition of both ConA and the test compound. As Table 1 shows, the test compound in accordance with the present invention exhibited a strong ability to inhibit the ConA induced reaction of T-lymphocyte blast genesis. Inhibition of ConA-induced reaction of T-lymphocyte blast genesis by the compound of the present invention (trihydrochloride) Concentration (µg/ml) ³H-thymidin uptake (dpm ± S.D.) Inhibition (%) control (10) 129,616 ± 35,477 0 compound of the present invention (100) 30,214 ± 25,033 76.7 compound of the present invention (100) 788 ± 182 99.4 Test 2: Inhibition of LPS-induced reaction of B-lymphocyte blast genesis by the compound of the present invention (trihydrochloride) The uptake of ³H-thymidine by B-cell blasts was measured in accordance with the method used in Test 1, except that the inducer ConA was replaced by 100 µg/ml of LPS from E. coli . The percentage inhibition of blast genesis by the test compound was also determined by the same method as used in Test 1. As Table 2 shows, the compound in acccordance with the present invention was highly effective in suppressing the LPS-induced genesis of B-lymphocyte blasts. Inhibition of LPS-induced reaction of B-lymphocyte blast genesis by the compound of the present invention (trihydrochloride) Concentration (µg/ml) ³H-thymidin uptake (dpm ± S.D.) Inhibition (%) control (0) 234,356 ± 61,436 0 compound of the present invention (10) 38,842 ± 21,573 83.4 compound of the present invention (100) 396 ± 216 99.8 Test 3: Inhibition of Production of IgM antibodies against Sheep Red Blood Cells by the compound of the present invention (trihydrochloride) CDF 1 mice (6 - 10 Wk old, female) were immunized by intravenous injection of 1 x 10⁸ sheep red blood cells. The mice were intraperitoneally administered varying doses (see Table 3 below) of the test compound for a period of 3 days starting from the day following the intravenous injection. Four days later, spleen cells were isolated from the mice and the number of IgM plaque-forming cells was counted by a known method (Yata et al.; Gan to Kagaku Ryoho (Cancer and Chemotherapy), vol., 2, pp. 903 - 908, 1975). The percentage inhibition of IgM antibody production was calculated by (1 - B/A) x 100 wherein A is the count for a control group (given physiological saline) and B, for the treated group. As Table 3 shows, the compound in accordance with the present invention strongly inhibited the production of IgM antibodies. Inhibition of IgM antibody production against sheep red blood cells by the compound of the present invention (trihydrochloride) Dose (mg/kg/day) IgM plaque-forming cell count (cells/10⁶ spleen cells x 100 ± 100 ± S.D.) Inhibition (%) 0 (control) 1,454 ± 197 0 1 1,304 ± 188 10.3 5 632 ± 87 56.5 10 392 ± 87 73.0 25 149 ± 53 89.7 50 64 ± 24 95.5 Test 4: Inhibition of IgG antibody production against sheep red blood cells by the compound of the present invention (trihydrochloride) CDF 1 mice (6 - 10 wk old, female) were immunized by intravenous injection of 1 x 10⁸ sheep red blood cells. The mice were intraperitoneally administered 50 mg/kg/day of the test compound for a period of 4 days starting on the day following the immunization or 3 days after that day. Seven days after the sensitization, spleen cells were isolated and the number of IgG plaque forming cells was counted by a known method (Yata et al., supra ). As in Test 3, the percentage inhibition of IgG antibody production was determined. The results as shown in Table 4, from which one can see that in either of the two administration schedules, the compound of the present invention exhibited strong inhibitory action on IgG antibody production. Inhibition of IgG antibody production against sheep red blood cells by the compound of the present invention (trihydrochloride) Dose (mg/kg/day) Administration schedule days after sensitization IgG plaque forming cell count (cells/10⁶ spleen cells ± S.D) Inhibition (%) control (0) - 508 ± 89 0 compound of the present invention (50) 1 ∼ 4 17 ± 16 96.7 compound of the present invention (50) 3 ∼ 6 19 ± 15 96.3 Test 5: Inhibition of IgE Antibody Production Against Egg albumin by the compound of the Present Invention (trihydrochloride) BALB/c mice (7 wk old, female) were challenged by intraperitoneal administration of 10 µg of egg albumin together with 4 mg of aluminium hydroxide gel. Thereafter, the mice where intraperitonelly administered 50 mg/kg/day of the test compound in accordance with the schedules shown in Table 5. After the challenge, blood was drawn from the fundus of each mouse at 10-day intervals for serum collection. The antibody titer of IgE in the serum was determined by passive cutaneous anaphylaxis as follows. Fischer 344 rats (female, 10 wk old) were injected with 0.05 ml of a diluted serum into the dorsal skin. Four hours later, 1 mg of egg albumin and 0.5 ml of 1 % Evans blue were injected into the tail vein, and the final serum dilution that produced a blue spot of diameter of at least 5 mm 30 minutes after the intravenous injection was used as a measure of the antibody titer. The results are shown in Table 5, from which one can see that the compound strongly inhibited IgE production when it was administered right after the antigenic challenge. Inhibition of IgE antibody production against egg albumin by the compound of the present invention (trihydrochloride) Administration schedule (days after challenge) Adminstration times IgE antibody titer (dilutions ± S.D.) 10 days 20 days 30 days 40 days (control untreated) 0 110 ± 85 230 ± 234 440 ± 339 680 ± 497 1 ∼ 8 8 13 ± 33 60 ± 31 320 ± 0 320 ± 0 9 ∼ 16 8 120 ± 98 110 ± 106 320 ± 0 480 ± 392 1 ∼ 16 16 0 ± 0 8 ± 9 80 ± 120 200 ± 131 Dose: 50 mg/kg/day (i/p.) Test 6: Inhibition of Allokiller T-lymphocyte Genesis by the Compound of the Present Invention (trihydrochloride) 3 x 10⁷ P815 mastocytoma cells were administered intraperitoneally to C 57 BL/b mice (10 wk old). Twelve days later, spleen cells were isolated and used as effector cells (E). Target cells (T) which were ⁵¹Cr labelled P815 mastrocytoma cells were challenged by the effector cells at varying E/T ratios and the allokiller activity was assayed in terms of the percentage liberation of ⁵¹Cr. The compound of the present invention was administered for nine consecutive days starting on the day following the transplantation or a single administration was made on the 9th day. As shown in Table 6, the compound of the present invention inhibited appreciably the allokiller activity against the graft. Inhibition of Allokiller T-Lymphocyte Genesis by the compound of the present invention (trihydrochloride) Dose of the compound of the present invention (mg/kg/day) Adminstration schedule (days after transplantation) Percent liberation of ⁵¹Cr E/T ratio 5 10 20 0 (control) - 34 48 68 5 1 ∼ 9 6 8 17 50 1 ∼ 9 0 0 1 50 9 21 30 51 E/T ratio: Effector cell (spleen cell)/target cell (⁵¹Cr mastocytoma) Test 7: Inhibition of Rejection of Skin Allograft by compound of the Present Invention (trihydrochloride) Skin sections (5 x 10 mm) were peeled from the tails of SHR rats (female, 10 wk old) and transplanted to Fischer rats (female, 10 wk old) on the back. Varying doses of the compound of the present invention were administered intraperitoneally for a consecutive period of 10 or 20 days starting on the day following the transplantation. Complete necrosis of the graft was used as a measure of rejection and the inhibitory action of the compound was evaluated in terms of how long the rejection was postponed. As shown in Table 7, the inhibitory action of the compound was dose-dependent. The longer the period of administration, the greater the inhibitory action. Complete inhibition of the rejection was ensured throughout the administration period. No inhibition of the bone marrow was observed. As is clear from the above data, the compound of the present invention has strong inhibitory action on transplantation immune reactions without causing any side effects on the bone marrow. The compound also exhibits strong inhibitory action both on the IgE, IgM and IgG antibody production and on allergies, especially those associated with humoral immunity. The following Examples are provided for further illustrations of the present invention. Example 1 Thirty parts by weight of a hydrochloride of the compound of the present invention was mixed with purified water to make a total of 2,000 parts. The solution was passed through a Millipore filter of GS type for sterilization purposes. Two grams of the filtrate was put into 10 ml vials and freeze-dried to prepare injections each containing 30 mg of the hydrochloride of the compound per vial. Example 2 Granules An intimate mixture of 50 party by weight of a hydrochloride of the compound of the present invention, 600 parts of lactose, 330 parts of crystalline cellulose and 20 parts of hydroxypropyl cellulose was compacted with a Roller Compactor R , and ground into particles which were sieved to provide granules of a size between 16 and 60 mesh. Example 3 Tablets A mixture of 30 parts by weight of a hydrochloride of the compound of the present invention, 120 parts of crystalline lactose, 147 parts of crystalline cellulose and 3 parts of magnesium stearate was processed with a V-type pelletizing machine to produce tablets each weighing 300 mg.

Claims: Use of Spergualin of the formula or a pharmaceutically acceptable salt thereof for the preparation of a medicament for use as transplantation immunity suppressor and anti-allergic agent.

Inventors: ABE, FUMINORI, FUJII, AKIO, ISHIZUKA, MASAAKI, NAKAMURA, TERUYA, TAKEUCHI, TOMIO, UMEZAWA, HAMAO, PROF.
Applicants: MICROBIAL CHEMISTRY RESEARCH FOUNDATION
Application year: 1985
Main classification: A61K31
IPC codes: A61P37, A61K31