AGENT FOR THE PREVENTION OF VIRAL INFECTIONS

Abstract

The invention relates to medicine and veterinary medicine, and more specifically to pharmacology, and can be used to prevent viral infections caused be RNA viruses that have a lipid capcid. An agent for prevention viral infections comprises viral material from RNA viruses that have a lipid capcid and stabilized colloidal selenium at a 1:1 ratio. The viral material from RNA viruses has titres of 6.0-8.0 lg TCD.sub.50/ml. To obtain colloidal selenium having particle sizes from 10 to 15 nm the colloidal selenium is stabilized with polyethylene glycol, and for colloidal selenium having particle sizes from 20 to 40 nm, the colloidal selenium is stabilized with cysteine.

Claims

1. An agent for the prevention of viral infections, wherein the agent contains viral material from RNA-containing viruses with a lipid capsid; wherein the agent contains stabilized colloidal selenium.

2. The agent according to claim 1, wherein the ratio of the weight parts of the viral material to the stabilized colloidal selenium is 1:1.

3. The agent according to claim 1, wherein the viral material from RNA-containing viruses has a titer of 6.0-8.0 lg TCD50/ml.

4. The agent according to claim 1, wherein stabilized colloidal selenium has particle sizes of 10 nm-40 nm.

5. The agent according to claims 1 and 4, wherein the agent contains colloidal selenium with particle sizes of 10 to 15 nm stabilized by polyethylene glycol.

6. The agent according to claims 1 and 4, wherein the agent contains stabilized colloidal selenium with particle sizes of 20 to 40 nm stabilized by cysteine.

7. The agent according to claim 1, wherein the agent contains the stabilized colloidal selenium at concentrations of 6.0-6.2%.

Description

DETAILED DESCRIPTION

[0032] To obtain the viral material, the original strain of the virus (in particular, VN-96 virus strain of transmissible gastroenteritis, influenza A virus strain/Krasnodar/101/35/59 (H2N2), with a specific title, is taken and brought to the titer of 6.0-8.0 lg TCD50/ml.

[0033] For the prevention of influenza, a viral material with a titer of 8.0 lg TCD50/ml is preferred, and for the prevention of transmissible gastroenteritis, with a titer of 7.0 lg TCD50/ml.

[0034] Viral material is stored at −18° C. to −20° C.

[0035] The invention is illustrated by the following examples.

EXAMPLE 1

[0036] Example of preparation of colloidal selenium with a particle size of 10 nm-15 nm. 100 mg sodium selenite containing 40 mg selenium is taken and dissolved in 10 ml distilled water. 0.52 g hydrochloric acid hydrozine is taken and dissolved in 5 ml distilled water. 10 ml sodium selenite is added to the conical flask, and 5 ml hydrozine is immediately added.

[0037] The solution is mixed on a magnetic stirrer at 450 rpm without heating until an intense brick-red color appears (precipitation of amorphous selenium) for 10 minutes.

[0038] The flask is filled with 10 ml polyethylene glycol (PEG-200), and the mixture is heated to 150° C. with intensive stirring until the water completely evaporates (boiling ceases).

[0039] After the water has evaporated, a deflegmator is placed on the flask and the temperature is raised to 220° C. for 15-30 minutes. The mixture is cooled to room temperature.

[0040] The mixture is centrifuged at 4200 rpm for 20 minutes. The add-on is placed on dialysis against a pH 7.2 phosphate-salt buffer.

[0041] The resulting mixture is concentrated on a rotary evaporator with a vacuum at 70° C. and a rotation speed of 50 vol./min. (25 ml of the mixture is concentrated to 15 ml). Then a saline solution is added to the resulting sediment until a colloidal selenium concentration of 0.062 mg/ml (6.2%) is obtained.

[0042] The size of selenium particles determined using the LIBRA 120 electron microscope (Carl Zeiss, Germany) was 10 nm-15 nm.

EXAMPLE 2

[0043] Example of preparation of colloidal selenium with a particle size of 20 nm-40 nm. 0.001 M selenic acid (H.sub.2SeO.sub.3) is added drop by drop to 0.01 M L-cysteine solution with constant stirring at room temperature in a volume ratio of 1:1.

[0044] Then saline solution is added to the resulting sediment until a colloidal selenium concentration of 0.062 mg/ml (6.2%) is obtained. The size of selenium particles determined using the LIBRA 120 electron microscope (Carl Zeiss, Germany) was 20 nm-40 nm.

EXAMPLE 3

[0045] Example of preparation of colloidal selenium with a particle size of 100 nm-140 nm. This colloidal selenium was also used in further experiments to obtain a preventive agent to prove the achieved result efficacy.

[0046] 0.5 ml of 1 M hydrochloric acid hydrazine solution and 0.125 ml of 1 M sodium selenite (rapidly developing yellow-orange color) are added to 2 mL distilled water. Within 30 seconds, this solution is added to the milk serum protein.

[0047] The mixture is mixed for 1 hour. After the orange staining appears, the reaction is stopped with a 1M solution of sodium hydroxide, by adjusting pH to 7.62. The resulting solution is dialyzed against a 0.01 M phosphate-salt buffer, than the mixture is concentrated.

[0048] Then a saline solution is added to the resulting sediment until a colloidal selenium concentration of 0.060 mg/ml (6.0%) is obtained.

[0049] The size of selenium particles determined using the LIBRA 120 electron microscope (Carl Zeiss, Germany) was 100 nm-140 nm.

[0050] The concentration of colloidal selenium (6.0-6.2%) obtained in Examples 1-3 provides a stable state of the selenium substance with particle sizes of 10 nm-140 nm. It has been shown experimentally that a selenium concentration of less than 6.0% and more than 6.2% leads to a violation of the required selenium particle size or selenium coagulation.

EXAMPLE 4

[0051] The agent for the prevention of viral infections is prepared as follows.

[0052] 1 ml viral material is taken from the VN-96 strain of the pig transmissible gastroenteritis virus in a titer of 7.0 lg TCD50/ml, to which 1 ml stabilized colloidal selenium with a particle size of 20 nm-40 nm is added, the resulting mixture is mixed.

[0053] The finished agent for the prevention of viral infections is a red-brown liquid.

[0054] Similarly to the one described in Example 4, viral material is prepared from influenza A virus strains/Krasnodar/101/35/59 (H2N2).

EXAMPLE 5

[0055] Justification of the preventive properties of the claimed anti-flu drug.

[0056] In the experiment, 70 white mice were used, they were allocated in 7 groups (10 animals in each), of which 2 were the reference group and 5, the test groups. In the first reference group, the mice were not immunized. In the second reference group, mice were immunized subcutaneously with a drug containing 0.1 ml saline solution and 0.1 ml influenza A virus strain/Krasnodar 101/35/59 (H2N2)—with a titer of 8.0 lg TCD50/ml. The drug was administered twice with an interval of 14 days. In all test groups, the drug was also administered subcutaneously twice with an interval of 14 days between injections.

[0057] At the same time, in the first test group, mice were immunized with a drug containing 0.1 ml colloidal selenium with a particle size of 10 nm-15 nm, stabilized with polyethylene glycol (PEG), and 0.1 ml viral material from the influenza A virus strain/Krasnodar/101/35/59 (H2N2)—with a titer of 8.0 lg TCD50/ml.

[0058] In the second test group, mice were immunized with a drug containing 0.1 ml cysteine-stabilized colloidal selenium with a particle size of 20 nm-40 nm and 0.1 ml viral material from the influenza A virus strain/Krasnodar/101/35/59 (H2N2)—with a titer of 8.0 lg TCD50/ml.

[0059] In the third test group, mice were immunized with a preparation containing 0.1 ml colloidal selenium stabilized with milk serum protein with a particle size of 100 nm-140 nm and 0.1 ml viral material from the influenza A strain/Krasnodar/101/35/59 (H2N2)—with a titer of 8.0 lg TCD50/ml.

[0060] In the fourth and fifth test groups, mice were immunized with a preparation containing 0.1 ml cysteine-stabilized colloidal selenium with a particle size of 20 nm-40 nm and 0.1 ml viral material from the influenza A strain/Krasnodar/101/35/59 (H2N2), with a titer of 7.0 lg TCD50/ml (group four) and a titer of 6.0 lg TCD50/ml (group five).

[0061] 28 days after the first administration of the drug, all groups of mice were infected with the intranasally virulent strain A/Brisbane/59/07 (H1N1) at a dose of 2.0 lg TCD50/0.05 ml.

[0062] 72 hours after infection with the virulent strain, the mice were killed in accordance with the ethical principles of handling laboratory animals. The lungs of the mice were extracted and a 10% suspension was prepared in mortars with grated glass. 10-fold dilutions of this suspension were introduced into 9-day-old chicken embryos. After 48 hours of incubation of infected chicken embryos at 37° C. in the thermostat, the embryos were placed in the refrigerator at 4° C. for 18-24 hours.

[0063] Then the embryos were opened, allantois fluid was sucked out, and the titer of viruses in the lungs of each group of mice was determined using a hemagglutination reaction. The results are presented in Table 1.

TABLE-US-00001 TABLE 1 Infectious titer Antibody Preparation composition of strain A/ titer Viral material Brisbane/59/07 for the strain Mice from strain Stabilized (H1N1) in A/Krasnodar/ group A/Krasnodar/101/ colloidal the lungs of 101/35/59 name 35/59 (H2N2) selenium immunized mice vIFA 1 reference Non-immunized mice 4.5 — group 1 g TCD.sub.50/ 0.2 ml 2 reference with a titer of 8.0 saline in the 5.0 640 group 1 g TCD.sub.50/ml in the amount of 1 g TCD.sub.50/ amount of 0.1 ml 0.1 ml 0.2 ml 1 test group with a titer of 8.0 1 g With a particle 3.0 1280 TCD.sub.50/ml in the size of 10-15 nm 1 g TCD.sub.50/ amount of 0.1 ml in an amount of 0.2 ml 0.1 ml 2 test group with a titer of 8.0 With a particle 1.0 160 1 g TCD.sub.50/ml in the size of 20-40 nm 1 g TCD.sub.50/ amount of 0.1 ml in an amount of 0.2 3 test group with a titer of 8.0 With a particle 4.5 640 1 g TCD50/ml in size of 100-140 nm 1 g TCD50/ the amount of in an amount of 0.2 ml 0.1 ml 0.1 ml 4 test group with a titer of 7.0 With a particle 1.0 80 1 g TCD50/ml in size of 20-40 nm 1 g TCD50/ the amount of in an amount of 0.2 ml 0.1 ml 0.1 ml 5 test group with a titer of 6.0 With a particle 1.0 80 1 g TCD50/ml in size of 20-40 nm 1 g TCD50/ the amount of in an amount of 0.2 ml 0.1 ml 0.1 ml

[0064] Table 1 shows that the titer of the virulent strain A/Brisbane/59/07 (NSH1) in the lungs of non-immune mice (reference group 1) was 4.5 lg TCD50/0.2 ml.

[0065] The titer of the virulent strain A/Brisbane/59/07 (NSH1) in the lungs of mice immunized subcutaneously with a dose of the influenza A strain/Krasnodar/101/35/59 (H2N2) with a titer of 8.0 lg TCD50/ml did not differ much from that in the first reference group—5 lg TCD50/0.2 ml.

[0066] However, there was a marked decrease in the titer of the virulent a/Brisbane/59/07 strain (H1N1) in the lungs of mice immunized subcutaneously with the same dose of the influenza strain A/Krasnodar/101/35/59 (H2N2) in combination with colloidal selenium. At the same time, when immunized with a drug with a particle size of 10 nm-15 nm, the titer was 3 lg TCD50/0.2 ml, and when immunized with a drug with a particle size of 20 nm-40 nm, a thousand-fold titer decrease was observed.

[0067] There was no decrease in the infectious titer of the virulent strain A/Brisbane/59/07 (H1N1) in the lungs of mice immunized subcutaneously with a drug with the stabilized colloidal selenium particle size of 100 nm-140 nm.

[0068] Studies on the use of viral material from the influenza strain A/Krasnodar/101/35/59 (H2N2) with the titers 7.0 lg TCD50/ml and 6.0 lg TCD50/ml in combination with stabilized colloidal selenium with particle sizes of 20 nm-40 nm have shown (see Table 1) that the drug with such a component content does not significantly increase the titers of specific viral neutralizing antibodies (the titer is 80) but provides effective protection against infection.

[0069] The results of studies presented in Example 5 to determine the protection efficacy when infecting mice with a virulent influenza strain prove that the best protection efficacy is achieved by introducing a drug containing stabilized colloidal selenium with particle sizes of 20 nm-40 nm and a viral material with a titer of 6.0-8.0 lg TCD50/ml.

[0070] Further studies to justify effective protection against transmissible gastroenteritis were conducted using a preparation containing stabilized colloidal selenium with particle sizes of 20 nm-40 nm and viral material against transmissible gastroenteritis with a titer of 8.0 lg TCD50/ml.

[0071] Since Example 5 (see Table 1) demonstrated that the anti-influenza protection efficacy correlates with a reduced, rather than an increased content of specific virus-neutralizing antibodies to influenza, the protection efficacy against transmissible gastroenteritis was assessed by the specific virus-neutralizing antibodies to transmissible gastroenteritis in laboratory animals, in mice and guinea pigs, and not by direct infection.

EXAMPLE 6

[0072] Justification of the preventive properties of the claimed drug against transmissible gastroenteritis. 3 groups of Guinea pigs were formed to prove protection efficacy: 5 animals in each group.

[0073] One group of animals was a reference group, saline solution at 0.5 ml dose was administered to them only.

[0074] The first experimental group of animals was injected with viral material from the strain of transmissible gastroenteritis virus, VN-96, with a titer of 7.0 lg TCD50/ml in combination with a saline solution (0.25 ml viral material and 0.25 ml saline solution).

[0075] The second experimental group was injected with viral material from the strain of transmissible gastroenteritis virus, VN-96, with a titer of 7.0 lg TCD50/ml in combination with stabilized colloidal selenium (0.25 ml viral material and 0.25 ml colloidal selenium) with a particle size of 20 nm-40 nm, taken in a ratio of 1:1.

[0076] 28 days after administration of the drugs, the titer of specific virus-neutralizing antibodies in the neutralization reaction was determined. The result is shown in Table 2.

TABLE-US-00002 TABLE 2 Animal Properties of Titer of specific group the administered viral neutralizing name drug antibodies Reference Saline solution 0    1 test group Viral material from the 1:128 VN-96 strain, with a titer of 7.0 1 g TCD50/ml 2 test group Viral material from the VN-96 1:8  strain with a titer of 7.0 1 g TCD50/ml + stabilized colloidal selenium with particle sizes of 20 nm-40 nm

[0077] Table 2 shows that the introduction of a drug containing viral material from the VN-96 strain with a titer of 7.0 lg TCD50/ml in combination with stabilized colloidal selenium with particle sizes of 20 nm-40 nm reduces the titer of specific virus neutralizing antibodies from 1:128 to 1:8, i.e. by 16 times.

[0078] Thus, the claimed agent for the prevention of viral diseases in humans and animals caused by RNA—containing viruses with a lipid capsid enables to quickly prevent viral diseases at minimal cost (by a single or double injection), regardless of the virus serotypes (pathogen varieties) and its antigenic composition, by activating the completed phagocytosis of viruses without increasing the titer of specific.

[0079] The claimed agent expands the range of antiviral preventive drugs and enables to solve the problem of preventing human viral diseases, especially influenza, due to intracellular virus suppression.