RU2245142C1 - Immunomodulating agent - Google Patents

Abstract

FIELD: organic chemistry, medicine, immunology.

SUBSTANCE: invention proposes applying 2,4-dichlorophenoxyacetic acid tris-(2-oxyethyl)-ammonium salt as an immunomodulating agent. The newly found properties provide the development on its base medicinal agents for treatment of inflammatory, autoimmune and lymphoproliferative diseases.

EFFECT: valuable medicinal properties of agent, expanded assortment of agents of indicated designation.

4 tbl, 4 ex

Description

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

A number of drugs are known that modulate immune responses. Immunomodulators include substances that specifically affect the cellular and humoral immune systems, selectively suppressing or stimulating the immune response.

The heterocyclic immunomodulator levamisole (M.D. Mashkovsky, Medicines, M., Medicine, 1986, vol. 2, pp. 169-170) can perform the functions of an immunomodulator that can enhance a weak reaction of cellular immunity, weaken a strong one and not act on a normal one. Levamisole has a dose-dependent effect on the immune system and, for example, it should be used as an immunostimulant with caution, since when doses are exceeded, it is possible not immunostimulating, but immunosuppressive, and in some cases from small doses of levamisole. The use of levamisole can cause various side effects. Clinical trials have shown that in 30-40% of patients receiving levamisole, various complications were recorded: headache, sleep disturbance, allergic reactions, agranulocytosis, leukopenia. Modern knowledge about the pathogenesis of many diseases associated with impaired interclonal relationships due to the upset of Th1 / Th2 regulatory influences and the balance of the corresponding cytokines put forward requirements for the search for new immunoactive drugs. In this regard, drugs that exhibit a multidirectional effect on integral indicators, such as antibody formation and HRT, which affect either humoral or only cellular immunity and do not have toxic effects on the body during primary in vivo screening for ictactic animals. In order to expand the arsenal of synthetic agents with immunomodulating properties, it is proposed to use the tris- (2-hydroxyethyl) ammonium salt of 2,4-dichloro-phenoxyacetic acid as an immunomodulator. Tris- (2-hydroxyethyl) ammonium salt of 2,4-dichloro-phenoxyacetic acid has antioxidant properties (“Clinical efficacy and safety of cephalosporin antibiotics manufactured by LLC“ ABOLmed ”Sb. .

The essence of the invention lies in the fact that the immunomodulating properties of tris (2-hydroxyethyl) ammonium salt of 2,4-dichloro-phenoxyacetic acid were studied by the ability of this compound to affect the number of nucleated blood cells in mice (myeloactive properties), the number of IgM antibody-forming cells in vivo (primary humoral response), delayed-type hypersensitivity reaction (HRT) - (cellular immunity), to spontaneous and mitogen-stimulated proliferation of splenocytes in mice in vitro (antiproliferative properties).

We used healthy sexually mature animals — mice of hybrids (CBAxC57BL16) 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. Assessment of myeloactive properties of the compound.

In an experimental group of animals, the compound was administered at a dose of 1/10 LD 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 as the number of leukocytes in one ml of blood and as a percentage of the corresponding control.

Table 1
The effect of tri- (2-hydroxyethyl) ammonium salt of 2,4-dichloro-phenoxyacetic acid on the number of nucleated blood cells in intact CBF1 mice Group Dose 1/10 LD ₅₀ (mg / kg) Number of nucleated cells % to control The control 9.87 × 10 ⁶ / ml Compound 75 15.1 * +52 * significantly, P <0.05

As can be seen from the data in Table 1, the compound reliably has myelostimulating properties, i.e. significantly more than 20% causes an increase in the number of leukocytes (52%).

Example 2. Evaluation of the effect of the compound on the primary humoral response.

In an experimental group of animals, the compound was administered at a dose of 1 / 10LD ₅₀ intraperitoneally daily for 7 days in a volume of 0.5 ml. The control animals were injected with a solvent (water) in the same volume and mode. On the day of the last administration, the compounds of animals were immunized intravenously with ram erythrocytes (EB) at a dose of 0.5 × 10 ⁷ / mouse. The amount of IgM AOK in the spleen of mice was evaluated on the 4th day after immunization by the number of local hemolysis zones in a semi-liquid medium by the modified method (Cunningham, 1968). The results were expressed as the absolute amount of tgM AOK in the spleen.

table 2
The effect of tris (2-hydroxyethyl) ammonium salt of 2,4-dichloro-phenoxyacetic acid on the absolute amount of IgM-AOK in the spleen of mice Groups The number of lgM-AOK The control 1839 Compound 4135 * * P <0.05

As can be seen from the data in table 2, the compound reliably stimulates the primary humoral immune response.

Example 3. Evaluation of the effect of the compound on the cellular immune response.

In an experimental group of animals, the compound was administered at a dose of 1/10 LD ₅₀ intraperitoneally daily for 5 days in a volume of 0.5 ml. On the day of the last administration of the compound, the 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 the solvent of the compound (water). HRT response was evaluated by local HRT (Crowte, 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 percentages or absolute values.

Table 3
The effect of tris- (2-hydroxyethyl) ammonium salt of 2,4-dichlorophenoxyacetic acid on the delayed-type hypersensitivity reaction (cellular immunity) Groups HRT (%) 1. Control 47.7 2. Connection 35.2 * ^* significantly, P <0.05

As can be seen from the data of Table 3, tris- (2-hydroxyethyl) ammonium salt of 2,4-dichloro-phenoxyacetic acid reliably suppresses cellular immunity.

Example 4. Evaluation of the effect of the compound on spontaneous, Con A and PWM-induced proliferation of spleen cells.

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) or pacifier mitogen (PWM, Sigma). 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 μp of the solution was added to each well of the plate. At the end of the incubation, the cells were collected on glass fiber filters ("Flow Lab") using a Harvester apparatus ("Tritertek"). 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 diphenyl-oxazolylbenzene 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 4
The effect of tris (2-hydroxyethyl) ammonium salt of 2,4-dichloro phenoxyacetic 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 Con A-induced proliferation (2 μg / ml) PWM-Induced Proliferation (1 μg / ml) The control 3346 15072 7226 0.75 3226 14285 6659 7.5 3566 14543 5610 * 75 2628 11447 5346 5610 * significantly relative to the corresponding control, P <0.05

As can be seen from the data, the compound exhibits inhibitory properties on PWM-stimulated proliferation of intact mouse spleen cells.

Assessing the overall results, it can be noted that the properties of stimulating leukopoiesis and the primary humoral response of tris (2-hydroxyethyl) ammonium salt of 2,4-dichloro-phenoxyacetic acid can be attributed to immunostimulants. And by properties, suppress the cellular immune response, spontaneous and stimulated cell proliferation in vitro to immunosuppressants.

The given examples illustrate the presence of tris- (2-hydroxyethyl) ammonium salt of 2,4-dichloro-phenoxyacetic acid with immunomodulating properties, which will make it possible in the future to create drugs based on it for treating various immunocompromised conditions.

Claims (1)

The use of tris- (2-hydroxyethyl) ammonium salt of 2,4-dichloro-phenoxyacetic acid as an immunomodulator.