RU2756353C1 - Immunomodulating humic agent - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: group of inventions relates to the field of pharmaceuticals, namely to a humic agent exhibiting immunomodulatory activity and an application thereof as an immunomodulator. The humic agent exhibiting immunomodulatory activity is produced from leonardite, lignin, coal, peat, sapropel, by means of ultrasonic dispersion of the pre-ground raw materials in a mixture with water at a certain temperature and a certain pressure, followed by cooling the solution to room temperature and diluting with water until reaching a content of humic substances of 1 to 20% wt., wherein the humic substances include humic and fulvic acids and salts thereof, as well as hydroquinone in an amount not exceeding 3% wt. of the weight of the humic substances.

EFFECT: group of inventions ensures production of a humic agent exhibiting immunomodulatory activity, containing a wide range of humic substances, and containing no chemical impurities, usable in the field of medicine and pharmaceuticals.

2 cl, 3 tbl, 2 ex

Description

The invention relates to the field of medicine and pharmaceutics, namely to humic preparations, and can be used to correct disorders in the immune system in pathological conditions associated with a deficiency of the Th1-dependent immune response (chronic, sluggish and recurrent infectious and oncological diseases).

Humic acids, fulvic acids have a powerful effect on any living organism due to their rich composition. They contain a full set of amino acids, macro- and microelements, minerals, as well as naturally occurring polysaccharides, vitamins, peptides, fatty acids, polyphenols, ketones, catechins, etc. There are about 70 useful components in total. This rich composition explains the positive biological effects of humic acid (see https://fb.ru/article/288472/guminovyie-kislotyi-chto-eto-takoe-i-kak-oni-vliyayut-na-organizm).

It is known that humic acids are able to stimulate some functions of human neutrophils. Antiviral activity has been revealed for preparations based on humic and humic-like substances, for example, for the herpes simplex virus - HSV. Humic compounds can be used as microbiocides, prophylactic agents against the spread of HIV / AIDS. The cytotoxic and antiviral properties of humic acids and fulvic acids isolated from coal and peat were studied. Studies have shown that all studied compounds were low-toxic and had a sufficiently high inhibitory effect against HIV infection (AI POPOV, VN ZELENKOV, TV TEPLYAKOVA Biological activity and biochemistry of humic substances. Part 2. Medico- biological aspect (literature review) Bulletin of the Russian Academy of Sciences, 2016/5, pp. 9-15). It has been shown that Oxygumate increases the activity of Th-1 cells and reduces the production of cytokines by Th-2 cells [Mariette et al., 2002]. The observed stimulation of the proliferation of human lymphocytes was associated with an increase in the production of IL-2 and the expression of IL-2 receptors, together with a decrease in the amount of IL-10 under the action of Oxygumate [Joone et al., 2003]. In vivo, it has been shown that oral administration of humic substances improves the parameters of innate immunity in experimental animals: there is an increase in the antibacterial activity of blood serum, phagocytic activity, lysozyme activity and bacterial agglutination [Sanmiguel et al., 2016]. This makes humic substances potential products for the development of multi-target immunoactive microbicides.

Research in the development of new biologically active compounds has shown that humic substances of various origins have immunomodulatory and anti-inflammatory effects, which makes it possible to use them for the prevention and treatment of chronic dermatoses, atopic dermatitis, allergic rhinitis and other diseases accompanied by inflammatory and allergic reactions. The use of humic substances as immunomodulators is promising. (I.A. Savchenko et al. BIOLOGICAL ACTIVITY OF HUMIC SUBSTANCES: PROSPECTS AND PROBLEMS OF THEIR APPLICATION IN MEDICINE (REVIEW). Journal Medial, No. 1 (23) April, 2019, pp. 54-60, DOI: http: // dx .doi.org / 10.21145 / 2225-0026-2019-1-54-60).

The influence of humic acids on the human immune system is shown. The use of derivatives of humic acids stimulates T-lymphocytes of both helper and suppressor subpopulations. The general regulatory mechanism of immune homeostasis is enhanced under the influence of neurohumoral-humoral rearrangements in the body, especially the systems "hypotolamus-pituitary adrenal glands", "adrenal cortex-thymus-spleen-lymph nodes", which is accompanied by the restoration of autoregulation of the immune response with a tendency to normalize the disturbed cooperation cells (Poluyanova I.E. Biological activity of humic substances obtained from peat, and the possibility of their use in medical practice. International reviews: clinical practice and health, 2017, N4, p. 114122).

From the prior art, it is known the use of water-soluble humic acids, isolated from high-moor pine-sphagnum-cotton grass peat by extraction with sodium pyrophosphate, as an agent having immunomodulatory activity and stimulating the development of Th1-dependent reactions of the immune response (patent RU 2662094, 23.07.2018).

It is also known to use water-soluble humic acids, isolated from high-moor peat magellanicum, with a number average molecular weight of 4871.2 Da, a weight average molecular weight of 18755.3, a polydispersity of 3.9, and a median of 9558.3 Da, as a means of stimulating the development of Th1-dependent reactions. type of immune response by regulating the balance of activation of pro-inflammatory properties of macrophages secreting cytokines IL-1β, IL-12 and TNF-α, and anti-inflammatory properties of macrophages that produce IL-10 cytokines (patent RU 2716504, 03/12/2020). The disadvantage of the known solution is that it contains only a narrow fraction of humic acids and does not contain other active components of humic substances.

The objective of the present invention is to obtain a humic agent containing a wide range of humic substances and having immunomodulatory activity, without the use of chemical reagents.

The technical result of the claimed invention is to obtain an immunomodulatory humic agent containing a wide range of humic substances and not having chemical impurities, which can be used in the field of medicine and pharmacy.

The specified technical result is achieved due to the fact that the claimed humic agent having immunomodulatory activity contains water and humic substances obtained from humic raw materials, a selected group, including leonardite, lignin, coal, peat and / or sapropel, by ultrasonic dispersion at a temperature of 30 -80 ° C and a pressure of 0.05-0.8 MPa, while the mass of humic substances is from 1 to 20 wt.%, And humic substances include humic and fulvic acids and their salts, as well as hydroquinone in an amount not exceeding 3 wt% based on the mass of humic substances.

It is also proposed to use the obtained humic agent as an immunomodulatory agent.

To obtain the claimed agent, pre-crushed raw materials (leonardite, lignin, coal, peat, sapropel) were used mixed with water, which was placed in an ultrasonic unit. Placed in the ultrasonic unit, a mixture of humic raw materials with water was heated to 30-80 ° C, and when the required temperature was reached, ultrasonic treatment was performed at a pressure of 0.05-0.8 MPa. After sonication, the solution was cooled to room temperature. The resulting product was diluted with water to a humic content of 1 to 20 wt%.

The study of the composition of the obtained product was carried out by the GC-MS method on an analyzer "Chromatek", consisting of a gas chromatograph "Chromatek-Crystal 5000" and a liquid dispenser DAZH-2M. For the identification of derivatives, an automatic database for the search and identification of gas chromatography-mass spectrometry data NIST17MS Library was used.

Research conditions:

The results of the study are shown in table. 1

According to the above data, in addition to humic and fulvic acids, the composition of the claimed agent also includes phenolic derivatives, in particular hydroquinone, flavanoids (chrysin) and other active substances. Therefore, the claimed agent is characterized by a wide range of active substances.

Toxicity study

Determination of indicators of acute toxicity included experiments on mice. Animals were randomly assigned to groups by randomization. The criteria for the acceptability of randomization were the absence of external signs of diseases and the homogeneity of groups by body weight (± 10%). The drug was administered intragastrically in increasing doses according to Litchfield-Wilcoxon. The highest dose was limited to the maximum possible volume of drug administration. To study each dose of the drug, groups of 10 animals of different sex were used. The observation period was 14 days. With the introduction of the drug in doses of 4000-8000 mg / kg (for humic acid), the animals showed a change in the response to picking up, changes in respiration, motor activity, and muscle tone; in some animals, a change in the consistency of feces was observed. When the drug was administered at a dose of up to 4000 mg / kg (for humic acid), all animals survived, when the drug was administered at a concentration of 6000 mg / kg (for humic acid), 5 animals out of 10 died, and when the drug was administered at a dose of 8000 mg / kg ( for humic acid), 8 animals out of 10 died. The surviving animals tolerated intoxication satisfactorily and after the end of the drug action for 14 days of observation, no signs of delayed influence were noted. There were no signs of prolonged clinical intoxication. The dynamics of the body weight of the experimental animals did not differ from the control. At the end of the observation period - on day 14, the surviving animals were slaughtered in order to determine possible pathological changes after a single dose of the drug. Examination of the experimental and control groups showed that all the animals in them were normally well-fed, had the correct constitution, smooth and shiny hair, shiny mucous membranes of normal color, clean and tidy natural openings. Macroscopic examination of the internal organs did not reveal any peculiarities. The analysis of the values of the mass coefficients did not reveal any significant differences between the groups of animals that received different doses of the drug. Thus, the results obtained allow us to assume that the obtained drug can be attributed to the V class, i.e. practically non-toxic medicinal substances.

Study of the immunomodulatory activity of the claimed agent.

The immunomodulatory activity of the claimed humic agent was studied in linear C57BL / 6 mice (males / females) at the age of 8-10 weeks (Guidelines for conducting preclinical studies of drugs. 4.1., M .: Grif and K, 2013, pp. 64-79) ... The studies were carried out in accordance with Directive 2010/63 / EU of the European Parliament and the Council of the EU on the protection of animals used for scientific purposes, GOST 33215-2014 "Rules for the equipment of premises and organization of procedures when working with laboratory animals."

Lycopid and pyrogenal were used as controls in in vivo experiments, and muramyl dipeptide was used in vitro.

Macrophages were obtained from a suspension of peritoneal cells, for which the animals were killed by dislocation of the cervical spine, the abdominal cavity was washed with ice-cold isotonic sodium chloride (RF), the cells were precipitated, resuspended in culture medium, and their viability was assessed in a test with 0.1% trypan blue. In the experiments, we used suspensions containing at least 95% of viable cells. The cells (1.5-2.0 × 106 / ml) were placed in plastic Petri dishes, cultured for 2 h at 37 ° C (in an atmosphere of 5% CO ₂ and absolute humidity) in a complete culture medium RPMI 1640 (Sigma), 10% ETS (Hyclone), 20 mM HEPES (Sigma), 0.05 mM 2-mercaptoethanol (Sigma), 50 μg / ml gentamicin (Sigma), 2 mM L-glutamine (Sigma ")). After 2 hours, macrophages adhering to the plastic were collected, transferred to flat-bottomed 96-well plates (2.5-3.0 × 106 cells / ml) and cultured under the above conditions in the presence of the claimed humic agent (HS, 10 μg / ml) ; 1 μg / ml LPS (serotype O111: B4, "Sigma"); 10 μg / ml muramyldipeptide (MDP, N-acetylmuramil-L-alanyl-D-isoglutamine, Calbiochem). The production of monokines was stimulated by the addition of LPS (1 μg / ml), lymphokines - with concanavalin A (4 μg / ml) or the lakonos mitogen (5 μg / ml). After 24 hours from the beginning of cultivation, the supernatant was collected from the wells and the concentration of cytokines in it was measured by the enzyme-linked immunosorbent assay using test systems: IL-2, IL-4, IL-10, IL-12 and IFN-γ ("eBioscience"), TNF-α - ("Vector-Best").

The amount of cytokines produced by human peripheral blood mononuclear cells (Mn) was determined in cell supernatants. For this, liquid for cell separation "Histopaque-1077" ("Sigma-Aldrich") with a density of 1.077 was placed in test tubes, after which whole blood of healthy donors was carefully layered with the addition of heparin (10 U / ml). After a 15-minute centrifugation at 400 g, the cells that formed a ring on the density gradient were harvested, washed three times with cold RF, resuspended in culture medium, and the viability was assessed. Then Mn (2.5-3.0 × 106 cells / ml) was placed in a 96-well plate and GS (10 μg / ml) was added, the production of cytokines was stimulated by the addition of mitogens. After 24 h of incubation, a cell-free supernatant was collected, in which the amount of cytokines was determined by the enzyme immunoassay using test systems according to the attached protocols: IL-10 and IFN-γ (R @ D Systems), TNF-α - (Vector-Best ).

GS was injected into mice daily intraperitoneally for 10 days, and the indicators of cellular and humoral immunity were determined in them. In preliminary in vivo experiments, it was revealed that for the manifestation of the immunomodulatory effect, the optimal daily dose of the claimed GS is a concentration of 1 mg / kg of body weight, an analogue of LPS pyrogenal (Branch "Medgamal" FNITsEM named after NF Gamaleya, Russia) - 5 μg / kg in 0.1 ml of physiological solution (FR) and lycopid (JSC "Peptek", Russia) - 2 mg / kg. To induce a Th1-dependent type of immune response, 5 days after the start of GS administration, the animals were immunized with an intraperitoneal injection of sheep erythrocytes (5 × 107) in 0.1 ml of FR.

To assess the effect of GS on the cellular link of immunity, a delayed-type hypersensitivity reaction was used. For this, on the 5th day after immunization, the animals were given a second (permissive) injection of ram erythrocytes into the hind paw pad - "experimental paw" (108 ram erythrocytes in 0.05 ml of isotonic sodium chloride solution). The contralateral paw was injected with 0.05 ml of sterile isotonic sodium chloride solution ("control paw"). After 24 hours, the animals were sacrificed, both paws were cut off along the protrusion of the bone below the articulation of the fibula and tibia and above the heel joint, the local inflammatory response was assessed by the difference in the mass of the experimental and control paws.

The effect of HS on the humoral link of immunity was assessed by the number of antibody-forming cells (AOC) in the spleen and the titer of antibodies in the blood serum in the hemagglutination reaction (HA), expressed as log2. For this, animals receiving a course of GS were killed on the 5th day after immunization with sheep erythrocytes (at the peak of IgM-AOK and IgG-AOK, respectively), spleen was isolated and blood serum was collected.

The data obtained in the course of the study were processed using the Statistica 8.0 statistical software package. For each sample, the arithmetic mean (X), the arithmetic mean error (m), and the arithmetic mean deviation (σ) were calculated. The normal distribution was checked using the Shapiro-Wilk test. Comparison of sample means was performed according to Dunnett's test for comparing several experimental samples with one control sample in the case of a normal distribution or according to the Kruskall-Wallis test for k-unrelated samples (k> 2) and Dunn's test in the case of a distribution that differs from the normal distribution.

GS according to the invention, when cultured with intact MFs, significantly increased the concentration of IL-12, a key cytokine responsible for the efficiency of the T-cell immune response, from 0.166 ± 0.028 pg / ml in spontaneous control to 5.107 ± 0.612 pg / ml. (i.e. more than 30 times). When macrophages were cultivated without the addition of any stimulants (medium), no significant amounts of this cytokine were found in the supernatant, and LPS even reduced the index by 1.9 times. The activating effect of muramyldipeptide was also significant relative to the control and did not differ significantly from GS.

In addition, GS significantly, more than 3 times, increased the secretion of another proinflammatory cytokine IL-2 by mitogen-stimulated splenocytes of mice from 104.023 ± 16.957 pg / ml with Con A stimulation to 326.904 ± 2.940 pg / ml with the addition of GS. Also, under the influence of HS, the concentration of IFN-γ increased by 7.4 times from 2.857 ± 0.601 in the control to 21.141 ± 2.244 pg / ml.

When using another model (human peripheral blood monocytes), it was shown that, upon addition of the claimed HS, the TNF-α concentration sharply increased from 1.486 ± 0.92 pg / ml in the control to 94.205 ± 2.933 pg / ml both during cultivation with HS and with MDP up to 151.070 ± 11.263 pg / ml. The activating effect of GS was also preserved when assessing their effect on the production of IFN-γ by lymphocytes: the cytokine concentration increased 72.5 times from 0.042 ± 0.007 to 3.045 ± 2.004 pg / ml.

Evaluation of the effect of the studied substances on the secretion of IL-10 showed that GS does not affect the spontaneous production of cytokine, in contrast to MDP, which significantly increased it by 7.1 times. Moreover, the incubation of mononuclear cells with GS under conditions of LPS-modeled inflammation led to a significant 4.2-fold decrease in the cytokine concentration. GS did not enhance mitogen-stimulated production of IL-4.

Thus, it was experimentally established that GS according to the invention reduce the production of IL-10 against the background of stimulation of the production of key pro-inflammatory cytokines IL-2, IL-12, TNF-α and IFN-γ by lymphocytes by antigen-presenting cells. It should be noted that in its cytokine-stimulating effect, GS is significantly superior to the reference drug MDP.

In addition, it was shown that the course administration of the GS of the invention to animals (mice of the C57 / BL6 line) against the background of the development of a Th1-dependent immune response in them, induced by the introduction of sheep erythrocytes, led to the suppression of the HRT response, a marker reaction of the cellular Th1 immune response. Course introduction of the comparison drug - lycopid - increased the indicator by 1.3 times. The magnitude of edema with the use of GS and pyrogenal decreased by 1.9 compared with the control and 2.3-2.4 times relative to lycopid.

It has been experimentally established that the humic active substances that make up the claimed humic agent, with direct exposure, reduce the production of IL-10 against the background of stimulation of the production of key pro-inflammatory cytokines by antigen-presenting cells - IL-2, IL-12, TNF-α and lymphocytes - IFN-γ , both in mice and in humans (TNF-α and IFN-γ). Course introduction of the claimed humic agent reduces the intensity of the reaction of cellular immunity and enhances the indicators of humoral immunity. The humic substances of the claimed humic agent are activators of the inflammatory properties of macrophages according to the Th1-dependent type of immune response and can expand the arsenal of low-toxic herbal agents that can stimulate the immune response in infectious and inflammatory processes - acute, chronic, sluggish, recurrent, against the background of oncological diseases.

Thus, the claimed humic agent has a high biological activity, which is manifested in the ability to regulate the balance of pro-inflammatory and anti-inflammatory cytokines. Therefore, the claimed agent can be used as an immunomodulatory agent, which confirms the achievement of the claimed technical result.

Claims (2)

1. A humic agent with immunomodulatory activity, containing water and humic substances obtained from humic raw materials, selected from the group including leonardite, lignin, coal, peat and / or sapropel, by the method of ultrasonic dispersion of pre-crushed raw materials in a mixture with water at a temperature of 30 -80 ° C and a pressure of 0.05-0.8 MPa, after which the solution is cooled to room temperature and diluted with water until the content of humic substances is from 1 to 20 wt.%, While humic substances include humic and fulvic acids and their salts, as well as hydroquinone in an amount not exceeding 3 wt% of the mass of humic substances. 2. The use of a humic agent according to claim 1 as an immunomodulator.