RU2093162C1 - Agent showing immunostimulating effect - Google Patents

Abstract

FIELD: medicine, immunology. SUBSTANCE: invention proposes the use of zeolite-containing tuff - shyvyrtuine as an agent showing immunostimulating effect. The proposed agent can be used for recovery of the damaged immune homeostasis, in part, for treatment immunodefficient and acute infectious sicknesses. EFFECT: enhanced effectiveness of agent. 4 tbl, 15 dwg

Description

 The invention relates to medicine, in particular immunology, and can be used to restore impaired immune homeostasis, including the treatment of immunodeficiency and acute infectious conditions.

There is a large group of drugs that increase the overall resistance of the body or its nonspecific immunity (stimulants - caffeine, phenamine, eleutherococcus, etc. and vitamins retinol, ascorbic acid, vitamins of group B, etc.), as well as affecting specific immune reactions, for example, a number thymus extracts (thymalin, T-activin, thymoptin, vilosen) [1]
Among drugs that can stimulate immune processes and specifically activate immunocompetent cells (T and B lymphocytes), as well as additional immunity factors (macrophages, neutrophils, etc.), microbial and yeast preparations (prodigiosan, pyrogenal, etc.) are known [1]
The prototype of the claimed invention can be considered prodigiosan - a bacterial polysaccharide, which is an amorphous grayish-yellow powder [1]
However, the usually used daily dosage of prodigiosan often causes side effects in the patient: fever, headache, aching joints, leukopenia, diarrhea.

 The objective of the invention is the search for immunostimulating agents of natural origin, effective at low therapeutic doses, eliminating side effects and having a wider compared with known immunostimulants and a beneficial effect on all body systems, i.e. on the body as a whole.

 Natural zeolite-containing tuffs of various zoning are known, among which clinoptilolite rocks are of primary importance. The high clinoptilolite content (from 30 to 90%) in domestic zeolite-containing tuffs determines the similarity of the geochemical, adsorption, ion-exchange, catalytic and sieve properties of various varieties of zeolite-containing tuffs, of which the Shivirtuin-clinoptilolite-containing tuff of the Shivyrtuy deposit (Chita region) is a typical representative.

 Qualitative and quantitative characteristics of shivirtuin are presented in table. 1 (Feeding animals and zeolites: Guidelines for the study of natural zeolites in the feeding of farm animals. Kemerovo. 1990. P.5-6).

 The standards for the content of toxic trace elements in zeolite-containing tuffs are given in accordance with the technical conditions for zeolites [1]. Table 1 shows that the content of toxic trace elements in shivirtuin does not exceed the norm determined by the technical conditions for zeolite raw materials.

 The grinding fineness of the used zeolite-containing tuff was less than 1 mm, which also corresponds to the technical conditions, where it is indicated that the most effective grinding fineness of these tuffs is up to 1 mm.

 A unique combination of sorption, catalytic, ion exchange, sieve and other properties in zeolite raw materials provides a wide range of its industrial and agricultural applications.

 However, no data on the use of zeolite-containing tuffs as immunostimulants in immunodeficiency states and acute infectious diseases have been found in the current state of the art.

 The essence of the invention lies in the use of zeolite-containing tuff of the Shivyrtuiskoye field as a means with immunostimulating action. Schemes for the effect of shivyrtuin are shown in FIG. 1-15: FIG. 1 effect of shivyrtuin on the weight of the thymus in irradiated mice; FIG. 2 the effect of shivirtuin on the number of nucleated cells in the thymus of irradiated mice; FIG. 3 the effect of shivirtuin on spleen weight in irradiated mice; FIG. 4 the effect of shivirtuin on the number of nucleated cells in the spleen of irradiated mice; FIG. 5 - the effect of shivirtuin on the number of leukocytes in the peripheral blood of irradiated mice; FIG. 6 the effect of shivyrtuin on the number of nucleated cells in the spleen of irradiated mice on the 4th day after EB immunization; FIG. 7 the effect of shivyrtuin on the value of the humoral immune response to EB in irradiated mice; FIG. 8 - the effect of shivirtuin on the value of the integral index of immunodeficiency in irradiated mice; FIG. 9 - the effect of shivirtuin on the weight of the thymus after administration of hydrocortisone; FIG. 10 the effect of shivirtuin on the number of nucleated cells in the thymus of mice after administration of hydrocortisone; FIG. 11 the effect of shivyrtuin on the weight of the spleen of mice after administration of hydrocortisone; FIG. 12 - the effect of shivirtuin on the number of nucleated cells in the spleen of mice after administration of hydrocortisone; FIG. 13 the effect of shivirtuin on the number of peripheral blood leukocytes in mice after the administration of hydrocortisone; FIG. 14 - the effect of shivyrtuin on the number of nucleated cells in the spleen of mice treated with hydrocortisone on the 4th day after EB immunization; FIG. 15 the effect of shivirtuin on the size of the humoral immune response to EB in mice treated with hydrocortisone.

 To confirm the claimed use of zeolite-containing tuff of the Shivyrtuysky field (Shivyrtuin), studies were conducted on laboratory animals (white rats and mice).

 Example 1. Immunostimulating effect of shivyrtuin during immunization of animals with sheep erythrocytes.

 In the first series of experiments, the magnitude of the humoral immune response was evaluated using the method of immunization of white Wistar rats with sheep erythrocytes. The immune response was determined on the 5th day after immunization of animals (table. 2). The introduction of shivyrtuin into the diet of rats lasted 3 months.

 An analysis of the results showed that feeding shivirtuin to animals during subsequent immunization with sheep erythrocytes increases the immune response.

 Shivirtuin intake did not affect spleen mass in rats. In the group treated with shivyrtuin, a relative increase in the number of rosette-forming cells (ROCK) was noted compared with the control. When evaluating the results of determining IgM-producing cells, the same patterns were established as in determining ROC. The immune response for this indicator in rats consuming shivyrtuin was almost 2 times higher than in the control.

 Thus, the three-month introduction of shivyrtuin in the diet of rats produces an immunostimulating effect on the animal organism, causing a more pronounced than in the control group immune response to the introduction of sheep erythrocytes.

 Morphologically, a pronounced white pulp hypertrophy was observed in the spleen in animals treated with shivyrtuin with food.

 In the lymphoid apparatus, activation of macrophage elements not only in the germination centers, but also in the marginal parts of the follicles is noteworthy (Table 3).

 A similar morphological reaction is observed in the follicles of the lymph nodes (paratymic and mesenteric root). In the lymph nodes, in addition, there are morphological signs of activity of both T-dependent and B-dependent structures. In the B-dependent zones of the lymph nodes, lymphoid follicles are increased in volume and quantity, in the germination centers of which accumulation of blast-transformed cells is noted. The meat strands are in a state of hypertrophy due to an increase in the number of plasmacytic cells. In the T-dependent zones of the lymph nodes, as well as the spleen, there are morphological signs of an increase in the conjugation function in the immune response: an increase in the absolute volume of the nuclei of reticular cells and macrophages, a predominance of lymphocytes differentiating in the plasmacytic direction among lymphoid cells, and an increase in the number of macrophage outlets.

 Dense lymphocytosis is observed in the cerebral sinuses of the lymph nodes, which indicates an increased function of eliminating lymphocytes from the lymph nodes.

 A histological examination of the intestines of rats treated with shivirtuin revealed an increase in the specific volume of lymphoid tissue in their own intestinal plate. In all sections of the intestine, the number of lymphoid follicles is significantly increased. The main part of the follicles has a clearly differentiated germination center. In the latter, activation of macrophages is noted, which form typical macrophage rosettes together with adjacent lymphoid cells. The mantle zone of the follicles consists of densely aggregated small and medium lymphocytes.

 Thus, a positive effect was found in shivirtuin: stimulation of the immune system, which can be of great importance in the treatment of very many diseases.

 Example 2. The effect of shivirtuin on the morphological and functional parameters of the immune status of animals in experimentally created models of immunodeficiency in vivo.

 In experiments on the immunostimulating effect of shivirtuin, F1 hybrid mice (CBA x C57B1), females aged 2-4 months, were used. Animals were kept under standard vivarium conditions, shivyrtuin was added to the feed, replacing 5% of the feed weight.

 Two methods were used to create experimental immunodeficiency in mice: a single x-ray irradiation at a dose of 200 rad and a single intraperitoneal injection of hydrocortisone acetate at a dose of 1 mg / mouse.

To identify the effect of shivirtuin on the parameters of the immunodeficiency state, all animals were divided into 5 experimental groups):
intact mice;
control mice irradiated at a dose of 200 rad;
control mice injected with hydrocortisone;
“experimental” mice irradiated at a dose of 200 rad and fed with the addition of shivyrtuin;
"experimental" mice that received an injection of hydrocortisone and received food with the addition of shivyrtuin.

 The animals of the “experimental” groups (groups 4 and 5) were fed shivirtuin with food for three weeks, after which they and the control animals were either irradiated (groups 2 and 4) or hydrocortisone was introduced (groups 3 and 5). After irradiation or administration of hydrocortisone, the animals of the "experimental" groups continued to receive shivirtuin with food until the end of the experiment. Intact mice (group 1) were not exposed to any effects.

 The day of irradiation (or administration of hydrocortisone) was designated as day 0. On the 3rd, 10th and 17th day after the onset of immunodeficiency in intact, control and "experimental" mice (on the same day in animals of all groups) evaluated the studied parameters of the immune status. For this, part of the mice from each group were killed, determining the number of leukocytes in the peripheral blood, the weight of the thymus and spleen and the number of nucleated cells in these organs, and part of the mice were immunized with sheep erythrocytes, determining the number of antibody-forming cells in the spleen on the 4th day after immunization .

The results obtained in the control and "experimental" groups were expressed as a percentage of the initial level, taking the value of the studied parameters in intact animals (group 1) as 100%
Animals were slaughtered, taking blood from them and secreting the thymus and spleen. Lymphoid organs were weighed, after which cell suspensions were prepared from them and the number of leukocytes in peripheral blood and the number of nucleated cells in the thymus and spleen were calculated in the standard way in the Goryaev chamber.

The value of the humoral immune response was evaluated according to the Cunningham method (Cunningham AJ Szenberg A. // Immunology 1968. V.14, No. 3. P. 599-600), determining the number of antibody-forming cells (AOK) in the spleen of mice on the 4th day after intravenous immunization of their EB with a dose of 200 million cells. For this, spleen cells were suspended in 5 ml of medium 199 and the number of nucleated cells in the spleen was counted in a Goryaev chamber. The resulting cell suspension was diluted 10 times and mixed in different objects (0.5 ml each with a complement solution from guinea pig serum (1: 5 dilution) and 10% suspension of sheep erythrocytes. This mixture was poured into 0.18 glass chambers 0.24 ml, which were incubated in a thermostat at 37 ^o C for 45 minutes, after which, under a binocular magnifier, the number of erythrocyte lysis zones corresponding to spleen cells producing antibodies to EB was calculated in each chamber.

 For the statistical evaluation of the various compared samples, the nonparametric Wilcoxon-Mann-Whitney test was used.

 In the table. 4 and the figures (Figs. 1-15) show arithmetic mean values (M) and mean errors (m). The significance level of the differences was taken to be P <0.05.

 Statistical data processing was performed on a PC / XT personal computer using the StatGraphics program (USA).

 To create a radiation model of immunodeficiency in mice, single exposure of animals in a relatively small dose (200 rad) was used, which practically does not increase the mortality of mice, but at the same time causes a significant damage to the immune system, which is manifested in the mass death of radiosensitive immunocompetent cells (mainly thymus lymphocytes) and in the suppression of their functional activity. The effect of the selected dose of radiation on the studied indicators of the immune status at different times after irradiation is presented in table. 4. It can be seen that after 3 days in irradiated mice there is a significant decrease in the value of all investigated indicators of immune status as morphological (the weight of the thymus and spleen and the number of leukocytes in peripheral blood are reduced by 50–70% compared with intact animals, and the number of nucleated cells in the thymus almost 90%), and characterizing the functional activity of the immune system (non-specific proliferative response of spleen cells to immunization is halved, and the number of cells synthesizing specific immunoglobulins M to sheep erythrocytes, drops to 15% of the control). But due to the fact that a small dose of radiation was used, which did not cause complete emptying of the bone marrow and sharp inhibition of the hematopoiesis process, a noticeable increase in the weight and cellularity of lymphoid organs and restoration of the intensity of the immune response to EB were observed at a later date (10 and 17 days after irradiation). although some indicators of immune status remained significantly reduced until the end of the experiment.

 It was assumed that such a model of "small" immunodeficiency caused by the action of radiation will reveal the influence of the studied zeolite-containing tuff of shivyirtuin on the process of restoration of the immune system of radiation injury. The results of the experiments are shown in FIG. 1-8.

 The data obtained indicate that the introduction of zeolites in the feed of experimental animals (before and after irradiation) has a definite effect on the process of restoration of immunological parameters decreased by radiation.

 So, for example, the addition of shivirtuin to animal feed significantly increases the number of nucleated cells in the spleen of irradiated mice (Fig. 4), the number of leukocytes in the blood of animals on the 17th day after irradiation (Fig. 5). Similarly, the administration of shivyrtuin affects the size of the humoral immune response to sheep erythrocytes in irradiated mice (Fig. 7).

 Considering the data obtained, it can be noted that the effect of shivirtuin on the indicators of the immune status in the radiation model of immunodeficiency is characterized by a tendency to increase the studied parameters compared with irradiated animals. To illustrate this shivirtuin-related effect in FIG. Figure 8 shows the values of the integral index characterizing the degree of immunodeficiency in irradiated mice that received and did not receive shivyrtuin with food at different times after the start of the experiment (the integral index of immunodeficiency is calculated by simple summation and averaging of all determined parameters expressed in relative units, with 1 taken the value of the corresponding parameter in intact animals). It can be seen that by the 17th day after irradiation, the index in the group of animals treated with shivyrtuin is higher than the value of this index in irradiated mice that did not receive it.

 In order to check how the effect of shivirtuin depends on the specific conditions created by the radiation model of immunodeficiency, the effect of shivirtuin on the parameters of the immune status in another standard model of immunodeficiency caused by the introduction of hydrocortisone in animals was experimentally studied. Moreover, the general experimental design (the studied parameters, the timing of their determination, etc.) was exactly the same when studying both models of immunodeficiency.

 From the data given in table. 4, it is seen that intraperitoneal administration of hydrocortisone to mice is accompanied by a decrease in the main measured indicators of immune status, and the severity of immunodeficiency on the 3rd day after exposure is close to that observed after irradiation at a dose of 200 rad. As in the radiation model, at a later date these indicators return to normal, and on the 10th and 17th days the magnitude of only some parameters significantly differs from their value in intact animals. The only (but important) difference between these models of immunodeficiency is that the administration of hydrocortisone not only did not reduce the intensity of the humoral immune response to ram red blood cells, but, on the contrary, led to a significant and very significant increase in the specific response to the antigen. Such a paradoxical increase in the intensity of the immune response in the presence of undoubted (tested by other indicators) immunodeficiency in animals can be explained by the well-known fact that T-suppressors, which specifically suppress the development of immune systems, are most sensitive to the inhibitory effect of hydrocortisone reactions (Behrens TW Goodwin JS // The pharmacology of lymphocytes. BHNY 1988. P. 425-439). Therefore, under certain experimental conditions, the effect of hydrocortisone, which suppresses suppressor activity to a greater extent than the functions of other cell populations, manifests itself in a visible increase in the intensity of immune responses (Ibid; Piccolella E. et. Al. // J. Immunol. 1985. V. 134, No. 2. -P.1166-1171). With a high degree of probability, we can assume that a similar stimulating effect of the relatively small dose of hydrocortisone used in the experiment on a specific immune response was also observed in the above experiments. This explanation of the results is confirmed by the fact that in the same animals, hydrocortisone suppressed the nonspecific proliferative response of splenocytes to antigenic stimulation, significantly reducing the number of nucleated cells in the spleen after immunization.

The results of a study of the effect of shivirtuin on immune status parameters in mice after administration of hydrocortisone are presented in FIG. 9-15 and basically come down to the following:
the addition of shivirtuin to the feed leads to a significant (60%) increase in spleen cellularity on the 17th day after the administration of hydrocortisone (Fig. 11);
under the influence of shivirtuin, on the 17th day of the experiment, the number of leukocytes in the peripheral blood increases in comparison with animals that did not receive shivirtuin;
shivyrtuin on the 10th day after the administration of hydrocortisone increases the number of cells in the spleen after immunization with sheep red blood cells (Fig. 14).

 In general, the data obtained indicate that, as in the radiation model of immunodeficiency, the oral administration of shivyrtuin to animals has a stimulating effect on the parameters of the immune status of mice at various times after the administration of hydrocortisone.

 It can be assumed that the most likely and justified, from the point of view of the data currently available, mechanism of action of shivyrtuin in immunodeficiency states is the binding on its surface and removal of bacterial toxins from the intestine, which play an important role in the conditions of artificially created models of immunodeficiency.

Claims (1)

 The use of natural zeolite-containing tuff of the Shivyrtuisky deposit of Shivyrtuin as a means with immunostimulating effect.

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