RU2253444C1 - Immunomodulator - Google Patents

Abstract

FIELD: drugs, medicine.

SUBSTANCE: invention relates to application of 2-chlorophenoxyacetic acid tris-(2-hydroxyethyl)ammonia salt, which represents known herbicide, as immunomodulator. Present invention makes it possible to produce drugs for mieloactivity stimulation and hypersensitivity inhibiting when treatment of infections, inflammation, autoimmune and lymphoproliferative disorders.

EFFECT: new drug for mieloactivity stimulation and hypersensitivity inhibiting.

3 tbl, 3 ex

Description

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

Recently, much attention has been paid to the development and study of specific agents that stimulate or suppress the body's immune responses. Existing immunotropic drugs do not have sufficient selectivity, and their use may be accompanied by severe side effects. 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 the violation of the interclonal relationships of T-helpers of the 1st and 2nd types and the balance of their corresponding cytokines put forward requirements for the search for new immunomodulating drugs. In this regard, drugs that exhibit a multidirectional effect on the integrated indices of antibody formation and HRT during primary in vivo screening for intact animals, affecting either humoral or cellular immunity only and not toxic to the body.

The aim of the invention is to expand the arsenal of synthetic agents with immunomodulating properties.

To achieve this goal, it is proposed to use tris- (2-hydroxyethyl) ammonium salt of 2-chlorophenoxyacetic acid as an immunomodulator.

Tris- (2-hydroxyethyl) ammonium salt of 2-chlorophenoxyacetic acid has antioxidant properties (Clinical Efficacy and Safety of Cephalosporin Antibiotics manufactured by ABOLmed LLC, Proceedings of NIIKI, ASMU and ICD, Novosibirsk, 2002, p. 179).

The essence of the invention lies in the fact that the immunomodulating properties of tris- (2-hydroxyethyl) ammonium salt of 2-chlorophenoxyacetic 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 (AOKs) in vivo (primary humoral immune response), to the HRT reaction (cellular immunity), to spontaneous and mitogen-stimulated proliferation of splenocytes in mice in vitro (immunoproliferative properties).

We used healthy, sexually mature animals of 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.

The LD ₅₀ for the compound upon intraperitoneal administration is 1500 mg / kg. 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 in the same volume and mode were injected with solvent (water). 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 Effect of Tris- (2-hydroxyethyl) ammonium salt of 2-chlorophenoxyacetic 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 150 13.3 × 10 ⁶ / ml * +34 * significantly, P <0.05

As can be seen from the table. 1, the compound reliably has myelostimulating properties, i.e. more than 30% causes an increase in the number of white blood cells in peripheral blood.

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

The compound at a dose of 1/10 LD ₅₀ (150 mg / kg) in a volume of 0.5 ml of solvent was administered intraperitoneally once a day daily for 7 days and at a dose of 1/100 LD ₅₀ (15 mg / kg) in a volume of 0, 5 ml of the solvent was administered 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.

table 2 The absolute amount of IgM AOK in the spleen of intact CBF1 mice under the influence of the course administration of tris- (2-hydroxyethyl) ammonium salt of 2-chlorophenoxyacetic acid Groups Dose 1/10 LD ₅₀ (mg / kg) IgM AOK / Spleen. Dose 1/100 LD ₅₀ (mg / kg) IgM AOK / Spleen. The control 1839 1839 Compound 150 7689 * fifteen 1739 * significantly, P <0.05

As can be seen from the data in Table 2, the tris- (2-hydroxyethyl) ammonium salt of 2-chlorophenoxyacetic acid exhibits a pronounced significant stimulating effect on the primary humoral response at a dose of 150 mg / kg.

Example 3. Evaluation of compounds for delayed-type hypersensitivity (HRT) in vivo.

Compounds at a dose of 1/10 LD ₅₀ and 1/100 LD ₅₀ in a volume of 0.5 ml were 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 percentages or absolute values.

Table 3 The effect of course administration of the compound at a dose of 1/10 and 1/100 LD ₅₀ on delayed-type hypersensitivity in intact CBF1 mice. Connection cipher Dose 1/10 LD ₅₀ (mg / kg) HRT (%) Dose 1 / 100LD ₅₀ (mg / kg) HRT (%) The control 47.5 47.5 Compound 150 40,4 fifteen 37.0 * * significantly, P <0.05

As can be seen from the data in Table 3, the compound reliably suppresses HRT at a dose of 1/100 LD ₅₀ - 15 mg / kg, i.e. significantly suppresses T-cell immunity.

Example 4. Evaluation of the immunoproliferative 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 at 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 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-chlorophenoxyacetic 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 3305 15140 6896 1,5 3486 14295 7258 fifteen 3056 15290 6453 150 2036 * 14034 * 5308 * * reliably appropriate control, P <0.05

As can be seen from table 4, the compound is able at a dose of 150 μg / ml to have a significant inhibitory effect on the immunoproliferative activity (spontaneous and mitogen-stimulated proliferation) of mouse spleen cells.

Assessing the overall results, it can be noted that the selective effect of the test compound on the body's immune responses: a stimulating effect on myeloid activity and a primary humoral response and a suppressive effect on delayed hypersensitivity, as well as on spontaneous and mitogen-stimulated proliferation of spleen cells of intact mice, allows to ascertain the presence of tris- (2-hydroxyethyl) ammonium salt of 2-chlorophenoxyacetic acid with immunomodulating properties, which may in future serve as again for the creation of new drugs intended for the treatment of infectious, inflammatory, autoimmune and lymphoproliferative diseases.

Claims (1)

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