RU2240794C1 - Immunomodulating agent - Google Patents

Abstract

FIELD: organic chemistry, medicine.

SUBSTANCE: invention proposes applying 2-methylindolylthioacetic acid tris-(2-hydroxyethyl)ammonium salt as an immunomodulating agent that is known early as a stabilizing agent of cellular membranes.

EFFECT: valuable biological property of agent, expanded assortment of agents of claimed designation.

3 tbl, 3 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

Currently, in the treatment of infectious-inflammatory, autoimmune and oncological diseases in clinical medicine, drugs that have immunomodulating properties are quite widely used (I.B.Mikhailov. Doctor’s handbook on clinical pharmacology. - Manual for doctors. - St. Petersburg. - 2001 . - 736 p.). However, it must be emphasized that existing immunomodulators do not have sufficient selectivity for action on a particular immunity link, and also often cause the development of undesirable side reactions.

For example, the heterocyclic immunomodulator levamisole known to doctors of various specialties (Kharkevich D.A. Pharmacology. - M. - 2001. - P. 485) is able to enhance weak reactions of cellular immunity, weaken strong ones and not act on normally expressed ones. Levamisole is characterized by a dose-dependent immunotropic effect, which is sometimes of an opposite nature (when using large doses of the drug, it is possible to observe the effect of immunosuppression instead of immunostimulation). In addition, the use of levamisole can cause serious side effects of a very diverse nature: neurological disorders, inhibition of blood formation, allergic reactions, etc.

Modern knowledge about the pathogenesis of many diseases, which is based on functional disorders of the cellular-humoral mechanisms of the formation of immune reactions, requires the search for new immunoactive drugs that are distinguished by their selective action on various parts of the immune response, as well as minimizing the risk of inducing side effects on the patient's body.

In order to expand the arsenal of effective and low-toxic agents with immunoactive properties, it is proposed to use Tris (2-hydroxyethyl) ammonium salt of 2-methylindolylthioacetic acid as an immunomodulator.

According to published data, Tris- (2-hydroxyethyl) ammonium salt of 2-methylindolylthioacetic acid is a stabilizer of cell membranes (Clinical efficacy and safety of cephalosporin antibiotics manufactured by ABOLmed LLC. - Proceedings of NIIKI, ASMU and MKB.- Novosibirsk, 2002. - S. 179).

The essence of the invention lies in the fact that the immunomodulating properties of tris (2-hydroxyethyl) ammonium salt of 2-methylindolylthioacetic acid were studied with respect to the regulation of proliferative activity of immunocytes (mitogen-stimulated proliferation of spleen cells in vitro), as well as with respect to the ability of this compound to influence humoral (the number of IgM - antibody-forming cells - IgM AOK) and cellular (HRT reaction) in vivo immunity.

We used healthy sexually mature animal mice - hybrids (CBAxC57BL / 6) Fl (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 using the nonparametric criterion U of Wilcoxon-Mann-Whitney.

Example 1. 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 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.

As can be seen from table 1, the test compound at a dose of 300 μg / ml significantly inhibited spontaneous and PWM-induced proliferation

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

The compound at a dose of 1/10 LD ₅₀ and at a dose of 1/100 LD ₅₀ (in a volume of 0.5 ml) was administered to mice intraperitoneally once a day daily 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 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 table 2, tris- (2-hydroxyethyl) ammonium salt of 2-methylindolylthioacetic acid at a dose of 300 mg / kg has a pronounced stimulating effect on the primary humoral response, while at a dose of 30 mg / kg, the stimulating effect is unreliable .

Example 3. The reaction of delayed-type hypersensitivity (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 (Crowie, 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 table 3, the compound significantly inhibits the cellular immune response (HRT) at a dose of 300 mg / kg (1/10 LD ₅₀ ).

The examples given illustrate the presence in Tris- (2-hydroxy-ethyl) ammonium salt of 2-methylindolylthioacetic acid of the ability to selectively affect immune responses (stimulation of humoral immunity, inhibition of cellular immunity and proliferative activity of spleen immunocytes). This allows us to attribute this compound to immunomodulators of multidirectional action and in the future to create drugs on its basis for the treatment of various immunocompromised conditions.

Claims (1)

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