RU2252757C1 - Immunomodulator - Google Patents

Abstract

FIELD: immunology.

SUBSTANCE: invention relates to application of 2-Br-phenoxyacetic acid tris-(2-oxiethyl)ammonium salt with antioxidant activity as immunomodulator. Claimed compound gas stimulates action on mieloactivity and original humoral response, which was estimated on the base of amount of lgM-antibody forming cells, as well as depressive action on cell immune response and cell proliferation of spleen. Compound of present invention with immunological properties is useful in production of pharmaceuticals for treatment of various immune pathological conditions.

EFFECT: new application of known antioxidant as immunomodulator.

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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.

The development and study of synthetic chemical compounds that stimulate or suppress the body's immune responses is one of the urgent problems of modern pharmacology and clinical immunology. The currently existing immunotropic drugs do not have a sufficient focus and selectivity of action, and their use may be accompanied by severe side effects.

The synthetic immunomodulator levamisole (M.D. Mashkovsky. Medicines. - Moscow, 2001 - T.2. - Page 194-195) can perform the functions of an immunomodulator - strengthen the weak, weaken the strong and not act on normal immune responses. Levamisole exhibits a dose-dependent effect - when using high doses, it is possible not an immunostimulating, but an immunosuppressive effect. The use of levamisole can cause various side effects. Clinical trials have shown that patients receiving levamisole have various complications: headache, sleep disturbance, allergic reactions, agranulocytosis, leukopenia, etc.

Considering that current knowledge about the pathogenesis of many immunopathological conditions underscores the importance of diverse disturbances in the interclonal relationships of cells of the immune system, the search for new chemical compounds exhibiting an opposite effect on the integral immunity indices during the initial in vivo screening of intact animals (antibody formation and HRT reaction), is an important area of modern experimental and clinical pharmacology.

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-Br-phenoxyacetic acid.

Tris- (2-hydroxyethyl) ammonium salt of 2-Br-phenoxyacetic acid and its analogues have antioxidant properties (Clinical efficacy and safety of cephalosporin antibiotics produced by 000 “ABOLmed”. Proceedings of NIIKI, AGMU and MKB. Novosibirsk, 2002. p. 179; www.abolmed.ru/pages/article33.htm).

The essence of the invention lies in the fact that the immunomodulating properties of tris (2-hydroxyethyl) ammonium salt of 2-Br-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 antibody-forming cells (IgM AOK ) in vivo (primary humoral immune response), on delayed-type hypersensitivity in mice (cellular immune response), on spontaneous and mitogen-stimulated proliferation of splenocytes in mice in vitro (antiproliferative CTBA).

We used healthy sexually mature animals: mice - hybrids (CBA × C57BL / 6) F1 (CBF1) of both sexes, 8–10-week-old with a body weight of 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 obtained were processed according to the nonparametric criterion U of Wilcoxon-Mann-Whitney.

Example 1. Determination of myeloactive properties.

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 of solvent daily for 7 days. 24 hours after the last administration, 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 tris (2-hydroxyethyl) ammonium salt of 2-Br-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 Control (n = 14) 9.87 × 10 ⁶ / ml Compound (n = 14) 200 12.0 × 10 ⁶ / ml * +21 * significantly, P <0.05; n is the number of animals

As can be seen from table 1, the compound at a dose of 200 mg / kg reliably has myelostimulating properties.

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

The compound in a dose of 1/10 LD ₅₀ and 1/100 LD ₅₀ in a volume of 0.5 ml of water was administered to experimental mice intraperitoneally once a day daily for 7 days. The control animals in the same volume and mode were injected with the solvent of the compound (water). 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 using the modified Cunningham method (1968). The results were expressed as the absolute amount of IgM AOK in the spleen.

As can be seen from the data in table 2, the test compound at a dose of 200 mg / kg shows a significant stimulating effect on the primary humoral response.

Example 3. 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 of water 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, 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) into the contralateral paw the solvent was added 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 3.
The effect of tris (2-hydroxyethyl) ammonium salt of 2-Br-phenoxyacetic acid on delayed-type hypersensitivity in intact CBF1 mice. Groups Dose 1/10 LD ₅₀ (mg / kg) HRT (%) Dose 1/100 LD ₅₀ (mg / kg) HRT (%) Control (n = 15) 47.5 47.5 Compound (n = 16) 200 29.4 * twenty 39.7 * P <0.05; n is the number of animals

As can be seen from table 3, the test compound at a dose of 200 mg / kg (1/10 LD ₅₀ ) significantly suppresses cellular immunity (HRT).

Example 4. 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) 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, a 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 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-Br-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 2 3372 15218 5874 * twenty 2711 12949 5584 * 200 2005 * 13596 4552 * * reliably appropriate control, P <0.05

As can be seen from table 4, the compound at a dose of 200 mg / kg (1/10 LD ₅₀ ) reliably inhibits spontaneous proliferation of spleen cells. The compound also in all doses used from 2 to 200 mg / kg reliably suppresses proliferation of spleen cells stimulated by the pituitary mitogen (PWM mitogen).

Assessing the results as a whole, it can be noted that the test compound has a selective effect on the body's immune responses: stimulating on myeloid activity and the primary humoral response, depressing on the cellular immune response and mitogen-stimulated proliferation of spleen cells. This allows us to state the presence of immunomodulating properties in Tris (2-hydroxyethyl) ammonium salt of 2-Br-phenoxyacetic acid, which suggests the possibility of creating new drugs on its basis for the treatment of various immunocompromised conditions.

Claims (1)

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