RU2224513C1 - Immunomodulating agent - Google Patents

Abstract

FIELD: medicine, pharmacy. SUBSTANCE: invention proposes using 2-methylphenylthioacetic acid tris-(2-hydroxyethyl)ammonium 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
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, drugs, 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, of interest are preparations that exhibit a multidirectional effect on the integral indicators of antibody formation and HRT during primary in vivo screening for intact animals, which affect either only humoral or only cellular immunity and do not have toxic effects on the body.

 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-methylphenylthioacetic acid as an immunomodulator.

 Tris- (2-hydroxyethyl) ammonium salt of 2-methylphenylthioacetic 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-methylphenylthioacetic acid were studied by the ability of this compound to affect the number of nucleated blood cells in mice (myeloactive properties), the delayed-type hypersensitivity reaction (HRT) - cellular immunity, to spontaneous and mitogen-stimulated proliferation of splenocytes in mice in vitro (antiproliferative properties), as well as to 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 leukocytes (by 55%).

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

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 (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 in Table 2, tris- (2-hydroxyethyl) ammonium salt of 2-methylphenylthioacetic acid reliably suppresses HRT.

 Example 3. 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 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 diphenyloxazolylbenzene per 1 liter 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; average data on triplet are presented.

 As can be seen from the data in Table 3, the compound exhibits pronounced dose-dependent inhibitory properties on mitogen-stimulated proliferation of intact mouse spleen cells.

 Example 4. Evaluation of antitumor activity.

It was carried out by a screening method in vitro on P 815 mast cells. The cells were seeded at a concentration of 10 • 10 ³ / well, incubated with the compounds for 24 hours, 6 µCi H ³ -thymidine was introduced 6 hours before the end of incubation. 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 tris- (2-hydroxyethyl) ammonium salt of 2-methylphenylthioacetic acid exhibits a significant antiproliferative effect.

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

 The above examples illustrate the presence of tris- (2-hydroxyethyl) ammonium salt of 2-methylphenylthioacetic 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-methylphenylthioacetic acid as an immunomodulator.

Images