RU2224512C1 - Immunomodulating agent - Google Patents

Abstract

FIELD: medicine, pharmacy. SUBSTANCE: invention proposes using 4-chlorophenylthioacetic acid dimethylethanolammonium salt as an immunomodulating agent. Early this agent is known as an anti-hemolytic and anti-aggregative agent. EFFECT: expanded assortment of agents of claimed 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.

 Recently, much attention has been paid to the development and study of specific agents that stimulate or suppress the body's immune responses. Currently existing drugs do not have sufficient selectivity of action and their use may be accompanied by severe side effects. They can have a depressing effect on blood formation and cause leukopenia, thrombocytopenia, anemia, pancytopenia, activation of a secondary infection, the development of septicemia are possible. This applies to almost all drugs that exhibit antitumor activity (cytostatics).

 There are drugs that are used, for example, as immunostimulants, but under certain conditions they can also manifest themselves as immunosuppressants.

 The heterocyclic immunomodulator levamisole (M.D. Mashkovsky, Medicines, M., Medicine, 1986, vol. 2, p. 169-170) can perform the functions of an immunomodulator that can enhance a weak response 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.

 Current 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 are of interest that exhibit a multidirectional effect on integral indicators, such as 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.

 In order to expand the arsenal of synthetic agents with immunomodulating properties, it is proposed to use 4-chlorophenylthioacetic acid dimethylethanolammonium salt as an immunomodulator.

 Dimethylethanolammonium salt of 4-chlorophenylthioacetic 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 dimethylethanolammonium salt of 4-chlorophenylthioacetic 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 immune response), on spontaneous and mitogen-stimulated proliferation of splenocytes in mice in vitro (antiproliferative properties), as well as on the proliferation of tumor cell lines (cytostatic properties).

 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).

 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 1/10 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.

 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 white blood cells (by 76%).

 Example 2. The determination of the number of IgM antibody-forming cells in vivo.

The compound at a dose of 1/10 LD ₅₀ in a volume of 0.5 ml was administered intraperitoneally once a day daily for 7 days and at a dose of 1/100 LD ₅₀ in a volume of 0.5 ml was administered intraperitoneally once a day for 5 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 animals were immunized intravenously with ram erythrocytes (EB) at a dose of 0.5 x 10 ⁷ / mouse. The amount of IgM AOK in the spleen of mice was evaluated 4 days 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 table. 2, 4-chlorophenylthioacetic acid dimethylethanolammonium salt reliably stimulates the primary humoral response.

 Example 3. Spontaneous, Con A and PWM-induced proliferation of spleen cells.

The spleen cells of mice were cultured in round-bottom immunological reaction plates ("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 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 imp. / min included thymidine per 2 • 10 ⁵ cells, presents the average data for the triplet.

 As can be seen from the data in Table 3, the compound is capable of exhibiting, at a certain dose, inhibitory properties on mitogen-stimulated proliferation of intact mouse spleen cells.

Example 4. Evaluation of the antitumor activity was carried out in vitro on cells of mast cell P 815. The cells were seeded at a concentration of 10 • 10 ³ / well, incubated with the compounds for 24 hours, 6 hours before the end of the incubation, 1 μCi H ³ -thymidine was added. At the end of the incubation, the cells were collected on glass fiber filters ("Flow Lab") using a Harvester apparatus ("Titertek). The results were expressed in imp./min. Included thymidine on 10 • 10 ³ cells, presents the average data for the triplet.

 As can be seen from the data in Table 4, the dimethylethanolammonium salt of 4-chlorophenylthioacetic acid exhibits a significant antiproliferative effect at a dose of 200 m kg / ml.

 Assessing the overall results, it can be noted that, due to the effect on the myeloid activity and the primary humoral response, the dimethylethanolammonium salt of 4-chlorophenylthioacetic acid can be assigned to immunostimulants, and to the mitogen-stimulated proliferation of spleen cells in intact mice and in vitro tumor cell proliferation.

 The given examples illustrate the presence of 4-chlorophenylthioacetic acid in the dimethiethanolammonium salt of 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 dimethylethanolammonium salt of 4-chlorophenylthioacetic acid as an immunomodulator.

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