RU2228175C1 - Immunostimulating agent - Google Patents

Abstract

FIELD: organic chemistry, medicine, pharmacy. SUBSTANCE: invention proposes using 4-chlorophenylsulfonylacetic acid tris-(2-hydroxyethyl)- ammonium salt as an immunostimulating agent that is known early as a stimulating agent for growth of microorganisms or as an inhibitor of aggregation of platelets. EFFECT: expanded assortment of agents of claimed designation. 3 tbl

Description

The invention relates to new biologically active substances from the class of derivatives of alkane carboxylic acids.

Known tris- (2-hydroxyethyl) ammonium salt of 4-chlorophenylsulfonyl acetic acid (G. G. Levkovskaya and other Binuclear derivatives of phenylthio-acetic acid. "Chemical and Pharmaceutical Journal", 1984, No. 4, S. 431-436), used as a growth promoter of barley malt (ed. St. USSR No. 1253973, C 07 C 147/08, 1986).

The aim of the invention is the creation of new effective immunoactive substances based on the class of alkane carboxylic acids, which include the tris- (2-hydroxyethyl) ammonium salt of 4-chlorophenylsulfonyl acetic acid.

The pronounced stimulating properties of tris- (2-hydroxyethyl) ammonium salt of 4-chlorophenylsulfonyl acetic acid with respect to the main immunity systems indicate the possibility of creating on its basis a new class of effective drugs - immunostimulants by identifying new, previously unknown properties.

To achieve the goal, a solution of tris- (2-hydroxyethyl) ammonium salt of 4-chlorophenylsulfonyl acetic acid of various concentrations was used, which was administered intraperitoneally to experimental animals daily for 5 days. On the day of the last administration, the animals were immunized (antigen was administered to stimulate the immune response). We used healthy sexually mature animal mice - hybrids (CBA × C57BL / 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 the 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 day (in the morning). The data obtained were processed by nonparametric criteria.

Example 1. The number of IgM and IgG antibody-forming cells (AOK) in vivo

An experimental group of animals was injected with tris (2-hydroxyethyl) ammonium salt of 4-chlorophenylsulfonyl acetic acid at a dose of 30 mg / kg (1/100 LD ₅₀ ) intraperitoneally daily for 5 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 of tris- (2-hydroxyethyl) ammonium salt, 4-chlorophenylsulfonyl acetic acid was immunized intravenously with ram erythrocytes (EB) at a dose of 0.5 × 10 ⁷ / mouse. The amount of AOK IgM 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 effect of tris- (2-hydroxyethyl) ammonium salt of 4-chlorophenylsulfonyl acetic acid was evaluated on the inductive phase of IgM antibody formation. To determine the IgG of AOK (Cunningham, 1968; Dresser, Wortis, 1965), primary immunization was performed intraperitoneally, and secondary immunization was carried out intravenously at a dose of 0.5 · 10 ⁷ EB / mouse. To assess the value of the humoral immune response, the amount of AOK IgG in the spleen of mice was calculated at the peak of the immune response by local hemolysis. Spleen cells were incubated for 2 h at 39 ° C in chambers with EB, guinea pig complement and rabbit antiserum against mouse IgG (2000-fold dilution). Hemolysis zones were counted at × 42 magnification.

As can be seen from the data in Table 1, the tris (2-hydroxyethyl) ammonium salt of 4-chlorophenylsulfonyl acetic acid stimulates the primary and secondary humoral immune response.

Example 2. The reaction of hypersensitivity delayed type (HRT) in vivo

The experimental group of animals, tris- (2-hydroxyethyl) ammonium salt of 4-chlorophenylsulfonyl acetic acid was administered at a dose of 30 mg / kg (1/100 LD ₅₀ ) intraperitoneally daily for 5 days in a volume of 0.5 ml. On the day of the last administration of tris- (2-hydroxyethyl) ammonium salt, 4-chlorophenylsulfonyl acetic acid was mice 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 tris- (2-hydroxyethyl) ammonium salt of 4-chlorophenylsulfonyl acetic acid (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.

As can be seen from the data table. 2, Tris- (2-hydroxyethyl) ammonium salt of 4-chlorophenylsulfonyl acetic acid stimulates HRT by 38%.

Example 3. Fc receptor-mediated macrophage phagocytosis

The phagocytic activity under the influence of tris- (2-hydroxyethyl) ammonium salt of 4-chlorophenylsulfonyl acetic acid was determined by spectrophotometric micromethod Fc receptor of mediated phagocytosis of sheep erythrocytes by macrophages (Rummage et al., 1985). The experimental group of animals, tris- (2-hydroxyethyl) ammonium salt of 4-chlorophenylsulfonyl acetic acid was administered intraperitoneally at a dose of 30 mg / kg (1/100 LD ₅₀ ) daily for 5 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 of the tris (2-hydroxyethyl) ammonium salt of 4-chlorophenylsulfonyl acetic acid, the animals were injected intraperitoneally with 10% peptone. On the 4th day, peritoneal exudate cells were isolated. Peptone-stimulated abdominal macrophages were cultured at a concentration of 2 · 10 ⁶ / ml for 24 hours in 24-well plastic plates in RPMI-1640 medium containing 10% fetal calf serum, 10 mM HEPES, 2 mM L-glutamine at 37 ° C in an atmosphere of high humidity with a content of 5% CO ₂ in the air. Then, 1 ml of 2.5% opsonized EB was added to the macrophage monolayer and cultured under the same conditions for 40 min. After that, unfaginated EBs were lysed with a hypotonic NaCl solution. To destroy macrophages and phagocytosis-induced EBs, 0.6 ml of 1% solution of sodium dodecyl sulfate (detergent) was added to each well, after which the amount of hemoglobin released from the destroyed EBs was determined spectrophotometrically using a MULTISCAN at a wavelength of 405 nm. Phagocytic activity was determined individually in each mouse.

As can be seen from the data in Table 3, Fc-mediated macrophage phagocytosis is stimulated by the action of the tris- (2-hydroxyethyl) ammonium salt of 4-chlorophenylsulfonyl acetic acid in comparison with intact animals.

Assessing the overall results, it can be noted that, in terms of their ability to stimulate the primary and secondary humoral immune responses, HRT, the phagocytic activity of macrophages of tris (2-hydroxyethyl) ammonium salt of 4-chlorophenylsulfonyl acetic acid can be attributed to immunostimulants.

Tris- (2-hydroxyethyl) ammonium salt of 4-chlorophenylsulfonyl acetic acid is non-toxic to warm-blooded animals and has a pronounced immunostimulating effect. Given the availability and low cost of tris (2-hydroxyethyl) ammonium salt of 4-chlorophenylsulfonyl acetic acid, it is promising for the creation of immunostimulating drugs.

Claims (1)

The use of tris- (2-hydroxyethyl) ammonium salt of 4-chlorophenylsulfonylacetic acid as an immunosuppressant.