RU2253448C1 - Immunosupressor - Google Patents

Abstract

FIELD: drugs, medicine.

SUBSTANCE: invention relates to application of 1-methylindolyl-3-thioacetic acid tris-(2-hydroxyethyl)ammonia salt, which is known hypolipidemic agent, as immunosuppressor. Present invention ales it possible to produce pharmaceuticals for transplanted organ and tissue rejection prophylaxis or treatment of various immune-associated diseases.

EFFECT: new drug for transplant rejection prophylaxis or treatment of immune-associated diseases.

3 tbl, 3 ex

Description

The invention relates to new biologically active substances from the class of arylheteroalkanecarboxylic acids and can be used in medicine and biology as the basis for the creation of drugs.

One of the leading mechanisms for the development of autoimmune disorders is the pathological activation of T-lymphocytes, accompanied by an increase in the production of growth factors for B cells, an increase in the synthesis of autoantibodies, and the development of immunocomplex disorders in various organs and tissues of the body.

Targeted inhibition of the functional activity of T-lymphocytes and other immunocompetent cells is extremely important for modern transplantology in order to prevent immune rejection of transplanted organs (kidneys, liver, heart) and bone marrow.

One of the drugs used to reduce the functional activity of T-lymphocytes for the treatment of autoimmune diseases and the prevention of transplant rejection is cyclosporin A (Russian Drug Register. - Issue 9. - 2002 - Page 969), which, being an effective immunomodulator It causes a number of serious side effects (nephro- and hepatotoxicity, arterial hypertension, anemia, fluid retention in the body, convulsive seizures, etc.), which greatly complicates its widespread use.

In order to expand the arsenal of synthetic agents with immunomodulating properties and not causing side reactions, it is proposed to use a new chemical compound as an immunosuppressant: tris (2-hydroxyethyl) ammonium salt of 1-methylindolyl-3-thioacetic acid.

This compound is a derivative of alkane carboxylic acids and has antiaggregant properties (“Clinical efficacy and safety of cephalosporin antibiotics manufactured by ABOLmed LLC, Proceedings of NIIKI, ASMU and MKB, Novosibirsk, 2002, p. 179).

The essence of the invention lies in the fact that the immunomodulating properties of tris- (2-hydroxyethyl) ammonium salt of 1-methylindolyl-3-thioacetic acid were studied by the ability of this compound to affect the number of nucleated blood cells in mice (myeloactive properties), delayed-type hypersensitivity in mice (T-cell immune response), to spontaneous and mitogen-stimulated proliferation of splenocytes in mice in vitro (antiproliferative properties against T- and B-lymphocytes).

Healthy sexually mature animals were used in the work: hybrid mice (CBAxC57BL / 6) F1 (CBF1) of both sexes, 8-10 weeks old, weighing 18-20 g. The spread in the groups according to the initial body weight did not exceed ± 10%. Control and experimental animals of the same age and obtained simultaneously from the same cattery ("Dawn", Tomsk). Before and during the experiment, control and experimental animals were kept in a vivarium under the same conditions: standard plastic cages with small wood shavings (no more than 10 individuals) on a standard diet. All studies were carried out at the same time of the day (in the morning). The data were processed using the nonparametric U Wilcoxon-Mann-Whitney test.

Example 1. Determination of myeloactive properties.

In an experimental group of animals, the compound was administered at a dose of 1 / 10LD ₅₀ intraperitoneally in a volume of 0.5 ml daily for 7 days, 24 hours after the last injection, the number of nucleated cells in the peripheral blood was determined. The control animals were injected with a solvent (water) in the same volume and mode. The criterion for assessing myeloactive properties is a change in the leukocyte formula of ± 20%. The results were expressed in the number of leukocytes in 1 ml of blood and in percentage to the corresponding control.

Table 1 The effect of tris (2-hydroxyethyl) ammonium salt of 1-methylindolyl-3-thioacetic acid on the number of nucleated blood cells in intact CBF1 mice Group Dose 1 / 10LD ₅₀ (mg / kg) Number of core
cells % to control The control 9.87 × 10 ⁶ / ml Compound 300 7.1 × 10 ⁶ / ml -22%

As can be seen from table 1, the compound, administered at a therapeutic dose of 300 mg / kg body weight for several days, has a pronounced myelodepressive effect.

Example 2. Evaluation of the effect of the compound on the cellular immune response.

The compound at a dose of 1/10 LD ₅₀ and 1/100 LD ₅₀ in a volume of 0.5 ml was administered intraperitoneally once a day daily for 5 days. The control animals in the same volume and mode were injected with a solvent of the compounds (water). On the day of the last administration of the compounds, mice were sensitized by intraperitoneal administration of 0.25% EB in a volume of 0.5 ml; on the 4th day after sensitization, a resolving dose of antigen was administered under the plantar aponeurosis of the right hind paw (50% EB in a volume of 50 μl). The solvent was injected into the contralateral paw in the same volume. The control animals in the same volume and mode were injected with a solvent of the compounds (water). HRT response was evaluated by local HRT (Crowle, 1975). The reaction was taken into account 24 hours after the introduction of the resolving dose of EB, the magnitude of the edema was evaluated with a caliper. The results were expressed as a percentage.

table 2 Effect of Tris (2-hydroxyethyl) ammonium salt of 1-methylindolyl-3-thioacetic acid on delayed hypersensitivity in intact CBF1 mice Dose 1/10 LD ₅₀ (mg / kg) HRT (%) Dose 1/100 LD ₅₀ (mg / kg) HRT (%) The control 47.5 47.5 Compound 300 35.0 * thirty 37.6 * * P <0.05

As can be seen from the table. 2, the test compound, administered to mice for several days at a dose of 30 and 300 mg / kg, significantly suppresses the HRT reaction, which mainly proceeds with the participation of T-lymphocytes.

Example 3. Evaluation of the antiproliferative properties of the compound.

The spleen cells of mice were cultured in round-bottom plates for immunological reactions ("Linbro") at + 37 ° C in an atmosphere of 5% CO ₂ and 95% air. The absolute number of cells introduced into the well was 200,000. The cells were stimulated with mitogens - concanavalin A (Con A, Sigma), which affects T-lymphocytes, or pacifier mitogen (PWM, Sigma), which affects B-lymphocytes. Mitogen concentrations were preliminarily titrated and used in the optimal dose, which was 2 μg / ml for Con A and 1 μg / ml for PWM. The compound in three doses was introduced into the wells simultaneously with mitogens. The proliferative activity of cells was evaluated by the inclusion of H ³ -thymidine in the DNA of dividing cells. The label was made 16 hours before the end of cultivation, 1 µCi in each well of the tablet. For this, the basic solution of H ³ -thymidine was first dissolved in RPMI-1640 medium to a concentration of 100 μCi / ml, and then 10 μl of the solution was added to each well of the plate. At the end of the incubation, cells were collected on glass fiber filters ("Flow Lab") using a Harvester apparatus ("Titertek"). The filters were dried and placed in scintillation counting bottles, and the radioactivity was counted in a toluene scintillator (4 g of diphenyloxazole, 0.1 g of diphenyloxazolylbenzene per 1 L of toluene) in a Delta liquid scintillation counter (USA). The results were expressed in pulses per minute of thymidine incorporated per 2 × 10 ⁵ cells; mean data on triplet are presented.

Table 3 The effect of tris (2-hydroxyethyl) ammonium salt of 1-methylindolyl-3-thioacetic acid on spontaneous and mitogen-stimulated proliferation of spleen cells of intact mice in vitro (cpm) The dose of the compound (μg / ml) Spontaneous proliferation ConA Induced Proliferation (2 μg / ml) PWM-induced proliferation (1 μg / ml) The control 3236 16010 7306 3 3040 15349 6559 thirty 2964 16681 6268 * 300 1068 * 5912 * 1349 * * reliably appropriate control, P <0.05

As can be seen from the data table. 3, the compound at a dose of 300 mg / ml reliably suppresses spontaneous and mitogen-induced proliferation of spleen cells (T and B lymphocytes). At a dose of 30 mg / ml, the compound also reliably suppresses proliferation of spleen cells stimulated by the pituitary mitogen (PWM mitogen).

Assessing the overall results, it can be noted that the selective depressive effect of the test compound on the body's immune responses (myeloactivity, T-cell immune response, spontaneous and mitogen-stimulated proliferation of T- and B-lymphocytes of spleen cells in vitro) allows the presence of tris- (2- hydroxyethyl) ammonium salt of 1-methylindolyl-3-thioacetic acid of immunomodulating properties of a suppressor nature, which makes it possible to create preparations based on it for the treatment of various autoimmune conditions, as well as to prevent the development of immune reactions aimed at rejection of transplanted organs and tissues.

Claims (1)

The use of tris (2-hydroxyethyl) ammonium salt of 1-methylindolyl-3-thioacetic acid as an immunosuppressant.