RU2177788C2 - Preparation to prevent and treat infectious diseases and correct pathological states - Google Patents

Abstract

FIELD: medicine; veterinary science. SUBSTANCE: the preparation necessary to prevent, treat and remove aftereffects of viral, bacterial, oncological, hepatic, gastro-intestinal, urogenital and immune system diseases, wounds, burns, stress contains polyprenol pyrophosphate of the general formula: H-[-CH₂-c(CH₃) = CH-CH₂-]_a-X, where X - pyrophosphorous acid anion, and a - 7, not less. EFFECT: suggested preparation is more active due to its polyfunctional activity at the cellular level. 5 tbl, 8 ex

Description

 The invention relates to means for the prevention, treatment and elimination of the consequences of viral, bacterial, oncological diseases, diseases of the liver, gastrointestinal tract, genitourinary system, immune system, wounds, burns, stress, used in medicine and veterinary medicine.

An antiviral agent is known, in the form of polyprenol monophosphate of the general formula H - [- CH ₂ —C (CH ₃ ) = CH — CH ₂ -] _a —X (polyprenyl phosphate), where a is the number of isoprene units, X is the anion of phosphoric acid (RU Patent 2005475, A 61 K 31/21, 1991). Using a known antiviral agent is not effective enough to fight viruses.

 The technical result from the use of the proposed biologically active substance is to increase the effectiveness of its antiviral effect.

The specified technical result is achieved by the use of polyprenol pyrophosphate [1, 2] of the general formula H - [- CH ₂ —C (CH ₃ ) = CH — CH ₂ -] _a —X, where X is the anion of pyrophosphoric acid, and at least 7, as a means for the prevention and treatment of infectious diseases and the correction of pathological conditions of a living organism.

 The proposed biologically active substance has multifunctional activity both at the cellular level and at the level of the organism as a whole.

 At the cellular level, it integrates into cell membranes, enhancing their conductivity, normalizes and activates the biosynthesis of cell surface glycoproteins, normalizes cell reproduction, intercellular and, as a result, interstitial interactions.

 In the body as a whole - it normalizes the functioning of the immune system, promotes tissue regeneration, improves the functioning of individual organs, enhances hematopoietic function.

 The described properties of the proposed biologically active substance make it possible to use it for the prevention, treatment and elimination of the consequences of viral, bacterial, oncological diseases, diseases of the liver, gastrointestinal tract, genitourinary system, immune system, wounds, burns, stress.

 The action of the tool can be illustrated by the following examples.

 Example 1. Antiviral activity of polyprenol pyrophosphate.

The experiments were carried out on mice of the C57B16 line infected intranasally with the influenza virus type A (H1N1) strain WSN at a dose of 5 LD ₅₀ .

 Simultaneously with the virus, polyprenol monophosphate or polyprenol pyrophosphate (0.4% solution at a dose of 5 μg per mouse) was administered once.

 The average life expectancy of animals that were injected with polyprenol monophosphate was 6.6 days, animals that were injected with polyprenol pyrophosphate - 8.1 days.

 The above example indicates a more effective effect of polyprenol pyrophosphate compared with polyprenol monophosphate.

 Example 2. Preventive protective effect of polyprenol pyrophosphate in mice treated with endotoxin of gram-negative bacteria.

 In the experiment, white mice weighing 20 g were used. Animals 1 day before endotoxin injection were injected intraperitoneally with polyprenol pyrophosphate at the rate of 100 mg per 1 kg of weight. Then, animals were injected intraperitoneally with 7 mg of Re chemotype glycolipid obtained from the bacteria Salmonella Minnesota R595 by extraction with chloroform and methanol. The death of animals is taken into account within 3 days. In each group - at least 20 animals. Without treatment with polyphrenol pyrophosphate, all mice die. Polyprenol pyrophosphate pretreatment protects 6 out of 20 animals from death. The differences between the groups are significant at p <0.01.

 Example 3. The use of polyphrenol pyrophosphate to correct the immune status in volunteers with dysbiosis.

 Tests were conducted on 3 volunteers with dysbiosis. Volunteers took orally 3 ml of a 0.4% solution of polyprenol pyrophosphate in the morning and evening for 3 days and 1 time on the fourth laziness. Before the first dose and 6 hours after the last dose, a blood and feces test was performed. In the blood, the content of IgG antibodies to the endotoxin of gram-negative bacteria (to the chemotype glycolipid Re) is determined, the total number of leukocytes and the number of leukocytes that bind endotoxin in the bloodstream or are able to bind endotoxin in vitro (in blood smears). The antibody content is determined using an enzyme-linked immunosorbent assay (ELISA) using protein A labeled with horseradish peroxidase. A pool of blood serum from 40 healthy volunteers was used as an indicator of norm. Leukocytes that bound endotoxin in vivo were detected by ELISA in blood smears using antibodies to Re-glycolipid labeled with horseradish peroxidase. To identify leukocytes capable of binding endotoxin in vitro, smears were first treated with Re-glycolipid and then conjugated to anti-glycolipid antibodies. The content of bacteria of various species in the feces was determined in a conventional manner by seeding on various nutrient media. The results of the study are shown in table 1.

 After repeated administration of the drug, all the studied parameters are restored to normal.

Example 4. Correction of pathological conditions. Correction of stress-induced immunodeficiency
To induce stress, experimental mice were kept under conditions of restriction of movement (muscle deprivation). For 10 days, the mice were placed one by one in plastic chambers 8.5 • 4.0 • 2.0 cm in size for 7 hours daily. Control mice were kept in standard cells of 10 individuals (standard cell size 42 • 14 • 11 cm). On the 10th day of stressful exposure, experimental (and control) animals were injected with a test antigen - sheep erythrocytes, after which the animals were kept under normal conditions [2]. To determine the level of stress, the method of local hemolysis in gel was used [1]. Mice were injected intraperitoneally with sheep erythrocytes (EB) 5 x 10 ⁸ in a volume of 0.5 ml. On the 5th day after immunization, spleens were taken from mice and erythrocyte suspensions were obtained. The tested cells (10 ⁶ in 0.1 ml) were introduced into molten agarose (0.7%), with freshly washed 20% sheep erythrocytes, after which the resulting mixture was layered on the surface of plastic Petri dishes with a diameter of 50 mm. After the mixture solidified, the cups were incubated at 37 ^° C. After 1 h, 1 ml of guinea pig complement was poured into the cups and incubated for an additional 1 h at 37 ^° C, after which the complement was drained and hemolysis zones were counted. The calculation of the number of antibody-forming cells (ATOC) was performed per 1 million viable splenocytes.

The following groups of mice were examined (10 animals per group):
1. Control - receiving EB (5%) intraperitoneally in 0.5 ml.

 2. Exposed to stress - receiving EB on the 7th day of stress exposure.

 3. Mice that were injected with polyphrenol pyrophosphate on the 1st day of stress exposure, and EB on the 7th day.

 The results presented in table 2 show that stress caused by hypokinesia induces in mice a significant suppression of the primary immune response to EB (from 204 to 70 ATOK per 1 million splenocytes). A single injection of the drug increases the number of ATOKs to normal, completely preventing the development of stress-induced immunodeficiency.

 Example 5. Correction of pathological conditions. The study of interferon production.

 The experiments were carried out on mice of the C57B16 line, surviving after infection with the influenza virus type A (H1N1) strain WSN.

Polyprenol pyrophosphate was intraperitoneally administered to C57B16 mice (mouse weight 12-14 g). After 24 hours, blood was taken from the experimental and control mice and the interferon status was determined by the following parameters: 1. The level of synthesis of interferon in the blood serum. 2. The level of interferon synthesis induced by Newcastle disease virus (virus induced interferon VBI, August ¹⁰ TTSD _50/02 mL). 3. The level of synthesis of interferon induced by staphylococcal enterotoxin A (mitogen-induced interferon, CEA, 1 μg / ml). Interferon titration is carried out in L-929 cell culture. As the test virus, 100 TCD ₅₀ of the mouse encephalomyocarditis virus are used. For the unit of interferon, the last dilution of the sample is taken, which provides 50% cell protection with complete destruction of the monolayer in the control.

 The following groups of mice were used in the experiment: 1) control (K), 2) control, treated with polyprenol pyrophosphate (CPP), 3) control, survivors of the viral infection (KB), 4) survivors of the viral infection and treated with polyprenol pyrophosphate (VPP) .

 It was established (Table 3) that polyprenol pyrophosphate restores the interferon status of mice surviving after a viral infection, while not affecting the interferon status of mice without interferon deficiency.

 Example 6. Correction of pathological conditions. Profile of cytokine mRNA.

The cell line MG-63 (human osteogenic sarcoma) was used. Cells were grown in Eagle MEM medium with 2 mM glutamine and 10% serum of cattle embryo (RED). When the cells reached a continuous monolayer, the growth medium was changed to a supporting one and the experimental cultures were treated with polyprenol pyrophosphate. After 2, 24 and 48 hours, the cells were removed with trypsin and versene solution, washed with physiological saline and total RNA was extracted (RNA extraction kits — Rneasy Total RNA System — Qiagen, Santa Clarita, CA) were used, followed by cDNA synthesis using oligo dT primers and AMV reverse transcriptase (New England Biolabs, Bedford, MA). cDNA was used as a template in the reaction of RT-PCR with primer pairs for the following cytokines - IFN-α, IFN-γ, IL-lb, IL-2, IL-4, IL-6, IL-10, TNF-α (Yamamura et al., 1991; Gelder et al., 1995; Lin et al., 1998). Reaction conditions - 94 ^o With 2 min; 35 cycles at 94 ^° C for 1 minute - 55 ^° C for 2 minutes - 72 ^° C for 2 minutes; 72 ^o With 5 min -> SHUT-OFF. The reaction product was detected by electrophoresis on a 3% agarose gel.

 In MG-63 cells, polyprenol pyrophosphate was shown to induce mRNA biosynthesis for IL-1 and IL-2. Induction was clearly observed 24 hours after treatment; the reaction product was not detected 2 hours after treatment; trace amounts of PCR product were detected 48 hours after treatment. It should be noted that mRNAs for IFN-α and TNF-α were constitutively represented in MG-63 cells.

 Example 7. Correction of pathological conditions. Effect on 2'-5'-oligoadenylate synthetase.

The enzyme 2'-5'-oligoadenylate synthetase (2-5A synthetase), in complex with dsRNA, polymerizes ATP into oligoadenylates, which activate ribonuclease, Rnase L. Activation of the 2-5A synthetase / Rnase L enzyme system enhances the decomposition of mRNA into cell and, as a result, to a decrease in the rate of cell reproduction, suppression of viral infection, etc. A transplantable line of human cells J-96 was used, obtained from the blood of a man suffering from subacute monocytic leukemia, and a transplantable line of murine fibroblasts L-929. Cells were cultured in medium 199 with 10% serum of cattle embryo. On the 2nd day of cultivation, when the cells reached an incomplete monolayer, the growth medium was replaced with a support medium (with 2% serum of cattle embryo) and treated with polyprenol pyrophosphate. After 24 hours, the cells were removed from the glass with Versen's solution, precipitated by centrifugation at 2000g for 5 min, suspended in buffer and frozen at -70 ^o C. After thawing the cells, the corresponding treatments were carried out and the activity of 2-5A synthetase was determined according to A. N. Narovlyansky and et al. (1988). The activity of 2-5A synthetase was expressed in moles of AMP included in the 2-5A dimer after 14 hours of incubation per 1 mg of cell lysate protein. As can be seen from table 4, the treatment of J-96 and L-929 cells with polyprenol pyrophosphate induced 2-5A synthetase. The activity of 2-5A synthetase increased to a level exceeding its original 5.7-fold for the J-96 cell culture and 2.9-fold for the L-929 cell culture.

 Example 8. Correction of pathological conditions. Effect on cAMP-dependent protein kinase.

Used a transplantable line of human cells J-96, obtained from the blood of a man suffering from subacute monocytic leukemia. Cells were cultured in medium 199 with 10% serum of cattle embryo. On the 2nd day of cultivation, when the cells reached an incomplete monolayer, the growth medium was replaced with a support medium (with 2% serum of cattle embryo) and treated with polyprenol pyrophosphate. After 24 hours, the cells were removed from the glass with Versen's solution, precipitated by centrifugation at 2000g for 5 min, suspended in buffer and frozen at -70 ^o C. After thawing the cells, the corresponding treatments and determination of the activity of the cAMP-dependent protein kinase were carried out according to A. N. Narovlyansky and et al. (1988). Protein kinase activity was expressed in pulses of 1 min per 1 mg of cell lysate protein.

 The data presented in table 5 allow us to compare the activity of this enzyme in the cell culture of J-96 without and with treatment with polyprenol pyrophosphate. It was shown that the activity of cAMP-dependent protein kinase is approximately 3.5 times higher in cell culture treated with polyprenol pyrophosphate.

Literature
1. Rip JW et al. , Distribution, metabolism and function of dolichol and polyprenols. // Prog. Lipid Res. - 1985. - v. 24. - 6.-p. 269-309.

 2. Shibaev V. N., Danilov L. L. New developments in the synthesis of phosphopolyprenols and their glycosyl esters. // Biochem. Cell. Biol. - 1992. - v, 70. - 6. - p. 429-437.

Claims (1)

The use of polyphrenol pyrophosphate of the General formula
H - [- CH ₂ —C (CH ₃ ) = CH — CH ₂ -] _a —X,
where X is the anion of pyrophosphoric acid,
a - not less than 7,
as a means for the prevention and treatment of infectious diseases and the correction of pathological conditions of the body.

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