RU2509801C2 - METHOD OF USING Bacillus intermedius RIBONUCLEASE - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and veterinary science. A method provides using bacterial 7P ribonuclease for increasing the effectiveness of suppression of RNA- and DNA-containing virus replication for treating acute viral diseases in mammals and warm-blooded animals.

EFFECT: Bacillus intermedius RNAase suppression of virus replication is ensured by using substantially lower doses of enzymes introduced into an infected body with the increased effectiveness of virus replication and therapeutic process.

2 cl, 8 tbl, 7 ex

Description

The present invention relates to the field of medicine and veterinary medicine. It can be used to suppress the multiplication of viruses, treat mammals and warm-blooded animals and humans, from diseases caused by RNA and DNA-containing viruses.

Ribonucleases (RNases) are RNA metabolism enzymes whose functions are to cleave mRNA, transform RNA precursors into mature forms, form small regulatory RNAs, and degrade certain types of RNA, including viral RNA.

It is known [1] the use of a drug with the commercial name "Endoglukin", consisting of an enzyme - endonuclease (bacterial), produced by the bacteria Serratia marcescens - in a mixture with the polysaccharide polyglucin. The disadvantage [1] is the significant limited scope of the drug - only for the prevention and treatment of viral diseases of bees and stimulate the development of bee colonies. To achieve other goals, the analog is not applicable. In addition, the bacterial endonuclease Serratia marcescens is unstable in an acidic environment and loses activity, which also limits the scope.

It is known [2] the use of the endonuclease of the bacterium Serratia marcescens as an antiviral drug for the recovery of potato from viral infection during microclonal propagation and the creation of transgenic forms of potato. The disadvantage [2] is the significant limited scope of the drug - only for the prevention and treatment of viral diseases of potatoes.

A common disadvantage of analogues [1] and [2] is that the tool "Endoglukin" and endonuclease Serratia marcescens are not suitable as a means of suppressing the reproduction of human and animal viruses.

Known are the results of a study of Actynomices rimosus ribonuclease as a possible means of inhibiting the development of human and animal viruses in vitro (in vitro) [3]. The disadvantage of the analogue [3] is that Actynomices rimosus ribonuclease is not suitable as a means of inhibiting the reproduction of viruses in animals.

The closest to the essence of the claimed invention, the prototype is a method of suppressing the reproduction of human and animal viruses by using the pancreatic RNase enzyme obtained from the pancreas of cattle (hereinafter referred to as the pancreatic RNase) [4].

The disadvantage of the prototype [4] is the need to use (to obtain a satisfactory result) a very high single dose of the enzyme introduced into the body. So, when evaluating the antiviral efficacy in newborn rabbits, the antiviral effect is achieved at an enzyme dose of 135 ... 200 mg / kg (10 mg / rabbit; rabbit weighing 50 ... 70 g). In mice, the effective dose is 250 mg / kg (5 mg / mouse; mouse weighing 20 g). For a person weighing 60 ... 70 kg for a single effective administration, 10 ... 15 g of the enzyme per dose will be required. A common disadvantage of using the prototype [4] for various warm-blooded living creatures is the need for specific use to obtain a positive result (suppressing the reproduction of human and animal viruses) -high doses of the enzyme (dose per unit mass of the experimental object). High doses of an enzyme introduced into the body - a foreign protein - lead to undesirable immunological complications (in the body). Complications essential for maintaining the health and even vitality of an organism exposed to a foreign protein.

The aim of the proposed invention is to increase the efficiency of suppressing the multiplication of viruses in the infected body, reducing the specific amount and concentration of the enzyme required to suppress the multiplication of viruses, increasing the effectiveness of the process of suppressing the multiplication of viruses, increasing the therapeutic effect.

The goals are achieved in that in order to increase the efficiency of suppressing the reproduction of viruses in the infected organism, for the treatment of viral diseases, a bacterial enzyme is used - RNase Bacillus intermedius, binase (hereinafter referred to as RNase Bi). Bacillus intermedius as a species based on analysis of 16S RNA according to the modern classification is classified as Bacillus pumilus (Gen Bank Accession No. HQ 650161.1). Structurally identical to the RNase of the original Bacillus intermedius 7P strain is used, the ribonuclease of other Bacillus intermedius strains and strains of other microorganisms. The term "other strains" means selected (cloned) and engineering strains.

The enzyme, which is the object of the invention, is, for example, the vital product of the known bacterium Bacillus intermedius, guanyl-specific RNase with a molecular weight of 12.3 kDa, 109 amino acid residues, pl = 9.5. Bacillus intermedius RNase was obtained from Bacillus intermedius culture fluid, for example, strain 7P, an extracellular alkaline RNase producer [4].

The antiviral activity of the claimed RNase Bi and RNase RV (prototype) is evaluated by well-known methods [6], for example, in vitro, and by intraperitoneal and / or intramuscular injection of the evaluated drugs into the animal. An example of the animals and / or their tissues most commonly used for this purpose (evaluating the effect of the test drug) is guinea pigs, pigs, chicken, and mouse.

The invention is illustrated by examples of its use in relation to the most typical viruses, for example, for influencing foot-and-mouth disease viruses (hereinafter referred to as VIA), Auezky disease virus (hereinafter referred to as BWA) and various strains of influenza virus. The above examples do not limit the scope and nature of the claims associated with them.

Example 1. Antiviral activity of the inventive RNase Bi and RNase RV (prototype) upon infection of a cell culture of transplantable kidneys of pig embryos (hereinafter referred to as PPES) in vitro (in vitro) with an RNA-containing foot and mouth disease virus (hereinafter referred to as VJ)

Evaluation of the antiviral activity of RNase Bi and RNase RV in vitro is as follows. A monolayer of a 48-hour cell culture of PPES in penicillin vials is pre-washed with a Hanks solution, after which they are infected with 0.1 ml of a suspension of VJ. Immediately after infection, in one part of the vials with the washed monolayer of PPES cells, the RNase of the pancreas diluted with growth medium (prototype) is added, in the other part of the vials, the claimed RNase Bi. The vials are left in a thermostat at plus 37 ° C for 1.0-1.5 hours and adsorb the virus to the test cells. After incubation, the monolayer of cells in vials with adsorbed virus is washed with Hanks solution. The Hanks solution after washing the infected monolayer of cells is poured into a collection vessel for infectious material for subsequent destruction. Then, in the same bottles with a monolayer washed from the non-adsorbed virus, add, for example, 2 ml of growth medium with the corresponding enzymes RNase RV or RNase Bi. Vials with a monolayer are again placed in a thermostat at + 37 °. The reproduction of the virus is judged by the appearance of a cytopathogenic effect, which is evaluated by crosses from 1 to 4 [5, 6]. The studied cells are destroyed, for example, by freezing and subsequent thawing. Material from experimental and control vials is titrated after freezing and thawing [5, 6]. For each dilution take 4 penicillin vials. The results are taken into account after 48 hours of infection with VJ and are expressed in TCD ₅₀ (tissue cytopathic dose causing destruction of 50% of the monolayer of infected cells calculated according to Reed and Mench) [5, 6]. In the tables, the numerical values of the TCD ₅₀ are expressed in decimal logarithm (calculated according to Reed and Mench, the numerical values of the TCD ₅₀ ), Table 1.

Table 1 Antiviral activity of RNase Bi (claimed) and RNase RV (prototype) in a culture of PPES cells infected with foot and mouth disease virus (VJ). TCD ₅₀ - tissue cytopathic dose causing destruction of 50% of the monolayer of infected cells Enzyme virus Enzyme dose
u / ml (mg / ml) The final titer of VYA Ig TCD ₅₀ RNase Bi + virus 100 TCD ₅₀ 60,000 units (0.08 mg / ml) 2.1 ± 0.24; P <0.01 RNase Bi 60,000 units (0.08 mg / ml) - RNase RV + virus 100 TCD ₅₀ 175000 units (0.58 mg / ml) 6.7 ± 0.32; P <0.15 RNase RV 175000 units (0.58 mg / ml) - Virus (control) - 7.5 ± 0.0

RNase Bi reduces the final titer of foot and mouth disease virus to Ig TCD ₅₀ = 2.1, RNase RV - to Ig TCD ₅₀ = 6.7 (decrease in titer of RNase Bi from control values is 5.4 Ig, RNase RV - 0.8 Ig). The efficiency of suppressing the reproduction of VW by RNase Bi without taking into account the differences in doses in activity and protein content is three times higher compared to pancreatic RNase (6.7: 2.1 = 3). The used dose of the claimed RNase Bi (60,000 units / ml or 0.08 mg / ml) is three times less than the dose of the prototype RNase RV (175,000 units / ml or 0.58 mg / ml) in activity (175,000 units: 60,000 units = 3) and seven times the protein content (0.58 mg: 0.08 mg = 7). The efficiency of suppressing the reproduction of VW by RNase Bi compared with RNase RV, taking into account the differences in the used doses, is 9 times higher in activity (175000 units: 60,000 units) × 3 = 9, for protein - 21 times higher (0.58 mg: 0, 08 mg) × 3 = 21.

Example 2. Antiviral activity of the inventive RNase Bi and RNase RV (prototype) upon infection of a cell culture of transplantable kidneys of pig embryos (hereinafter referred to as PPES) in vitro (in vitro) with DNA-containing virus of Auesca disease (hereinafter referred to as BWA)

Evaluation of the antiviral activity of RNase Bi and RNase RV in vitro is as follows. A monolayer of a 48-hour culture of PPES cells in penicillin vials is pre-washed with a Hanks solution, after which they are infected with 0.1 ml of a suspension of BWA. Next, perform the steps described in Example 1. The results are taken into account after 72 and 96 hours for infection with VBA and expressed in TCD ₅₀ calculated according to Reed and Mench [5, 6]. In the tables, the numerical values of the TCD ₅₀ are expressed in decimal logarithm (these numerical values of the TCD ₅₀ calculated according to Reed and Mench), Table 2.

table 2 The antiviral activity of RNase Bi (claimed) and RNase RV (prototype) in the culture of cells of PPES infected with Auezky's disease virus (WBA).
TCD ₅₀ is a tissue cytopathic dose that causes the destruction of 50% of the monolayer of infected cells. Enzyme virus Enzyme dose u / ml (mg / ml) The final titer of BWA Ig TCD ₅₀ * RNase Bi + virus 100 TCD ₅₀ 60,000 units (or 0.08 mg / ml) 2.00 ± 029
P <0.01 RNase Bi 60,000 units (0.08 mg / ml) - RNase RV + virus 100 TCD ₅₀ 175000 units (0.58 mg / ml) 3.46 ± 0.11
P <0.01 RNase RV 175000 units (0.58 mg / ml) - Virus (control) - 5.25 ± 0.23

RNase Bi reduces the final titer of BWA to Ig TCD ₅₀ = 2.0, RNase RV - to Ig TCD ₅₀ = 3.46 (the decrease in the titer of RNase Bi virus from control values is 3.25 Ig, RNase RV - 0.8 Ig ) The efficiency of the suppression of WBA multiplication by RNase Bi without taking into account the difference in doses in activity and protein content is four times higher compared to pancreatic RNase (3.25: 0.80 = 4). The used dose of the claimed RNase Bi (60,000 units / ml or 0.08 mg / ml) is three times less than the dose of the prototype RNase RV (175,000 units / ml or 0.58 mg / ml) in activity (175,000 units: 60,000 units = 3) and seven times the protein content (0.58 mg: 0.08 mg = 7). The efficiency of the suppression of WBA reproduction by RNase Bi compared with RNase RV, taking into account the differences in the used doses, is 12 times higher in activity (175000 units: 60,000 units) × 4 = 12, in protein - 28 times higher (0.58 mg: 0, 08 mg) × 4 = 28.

Example 3. Antiviral activity of the inventive RNase Bi and RNase RV (prototype) upon infection of mice with foot and mouth disease virus

Work with mice is performed according to a known technique [5]. Evaluation of antiviral activity in mice is as follows. Ten groups of white outbred mice weighing 18-20 g (in each group of 10 animals) are _taken , and 100 LD ₅₀ are administered intravenously (LD ₅₀ is a lethal dose that causes the death of 50% of animals) type A _{22-50 VW} adapted to these animals . After infection of the animals of the two control groups, an isotonic sodium chloride solution is administered, one group intramuscularly, the other intraperitoneally.

The four groups of infected animals are administered the inventive RNase Bi. Two of them injected the enzyme at a dose of 15,000 units or 0.02 mg / 1.0 g of the mass of mice, one group intraperitoneally, the second intramuscularly. The remaining two groups (mice) are injected with RNase Bi at a dose of 17,500 units or 0.023 mg / 1.0 g of the mass of mice, one group intraperitoneally, and the second intramuscularly.

Four groups of infected animals are injected with pancreatic RNase (prototype). Two of them (groups) inject the enzyme at a dose of 60,000 units or 0.2 mg / 1.0 g of the mass of mice, one group intraperitoneally, the second intramuscularly. The remaining two groups are injected with pancreatic RNase at a dose of 112,000 units or 0.35 mg / 1.0 g of the mass of mice, one group intraperitoneally, and the second group intramuscularly

Compared RNases are administered after infection twice a day, for 5 days. The antiviral effect of enzymes is judged by animal survival. The results are expressed as a percentage in relation to the total number of animals in the group (table 3).

The antiviral activity of RNase Bi at a dose of 15,000 units or 0.02 mg / 1.0 g of the mass of mice varies depending on the method of enzyme introduced from 25 to 50% of surviving animals. The life expectancy of animals that were injected with the enzyme, but which were not included in the group of survivors, varies from 3 to 6 days, depending on the method of administration of the enzyme.

The antiviral activity of pancreatic RNase at a dose of 60,000 units or 0.2 mg / 1.0 g of mouse weight, activity 4.0 times higher than the dose of RNase Bi (60,000 units: 15,000 units = 4), and the protein exceeds 10 times (0.2 mg: 0.02 mg = 10) with the intraperitoneal and intramuscular route of administration does not lead to the survival of animals.

With the intraperitoneal administration of RNase Bi at a dose of 17500 units or 0.023 mg / per 1 g of the mass of mice, 30% of the animals survive. RNase of pancreas with intraperitoneal administration of 112,000 units or 0.35 mg / 1 g of weight does not lead to the survival of animals.

With intramuscular injection of RNase Bi at a dose of 17,500 units or 0.023 mg / per 1 g of mass, 50% of infected animals survive, RNase of the pancreas (prototype) with intramuscular administration of 112,000 units or 0.35 mg / per 1 g of mass provides survival of 20% of animals. The antiviral effect of BiRNase Bi against VW without taking into account the difference in doses in activity and protein content is two and a half times higher compared to pancreatic RNase (50%: 20% = 2.5). The used dose of the claimed RNase Bi (17500 units / ml or 0.023 mg / ml) is six times less than the dose of the prototype RNase RV (112000 units / ml or 0.35 mg / ml) in activity (112000 units: 17500 units = 6) and 15 times less in protein content (0.35 mg: 0.023 mg = 15). The antiviral effectiveness of RNase Bi compared with RNase RV (prototype), taking into account the differences in the doses used, is 15 times more active (112,000 units: 17500 units) × 2.5 = 15, 37.5 times more for protein (0.35 mg: 0.023 mg) × 2.5 = 37.5.

Example 4. Evaluation of antiviral activity against foot and mouth disease in rabbits using RNase Bi other (in relation to the strain of Bacillus intermedius 7P) strain

Work with rabbits is performed according to the known method [5]. The assessment is carried out as follows. Ten groups of 10 newborns of 4-5 day old rabbits are taken - two control groups, eight others experimental ones.

Animals of one control group and four experimental groups are infected subcutaneously with lapinized (adapted to rabbits) VW strain “0” with an infectious virus titer of 5.5 1d TCD ₅₀ in 0.1 ml (the initial titer of the virus in sucker mice is 7.5 Ig TCD ₅₀ in 0.1 ml). Animals of the four experimental groups immediately after infection are injected with RNase. Two groups of animals RNase Bi at a dose of 25,000 units or 0.033 mg / 1.0 g of rabbit weight (one group intraperitoneally, the other intramuscularly). Instead of the enzyme, an isotonic sodium chloride solution is administered to the animals of the control group.

In two other groups, animals are injected with RNase RV at a dose of 100,000 units or 0.33 mg / 1.0 g of weight (in one group intraperitoneally, in the other - intramuscularly)

Animals of the second control group and four experimental groups are infected subcutaneously with lapinized (adapted to rabbits) VW strain “0” with a titer of 3.5 Ig infectious virus in 0.1 ml. Animals of the four experimental groups immediately after infection with the virus injected RNase. Two groups of RNase Bi 25000 units or 0.033 mg / 1.0 g weight (one group intraperitoneally, the other intramuscularly). Two other groups are injected with a prototype - pancreatic RNase of 100,000 units or 0.33 mg / 1.0 g of mass (in one group, intraperitoneally, in the other, intramuscularly).

RNases are administered 1 time per day during the 5-day observation period. Instead of the enzyme, an isotonic sodium chloride solution is administered to the animals of the control group. The antiviral effect of enzymes is judged by animal survival. The results are expressed as a percentage in relation to the total number of animals in the group (table 4).

Table 4 Antiviral activity of RNase Bi (claimed) and RNase of the pancreas (prototype) upon infection of newborn rabbits with foot and mouth disease virus.
In parentheses () is the time of death of control animals, which are injected with an isotonic solution of sodium chloride instead of the enzyme. In the control, infected with a virus with a titer of 5.5 Ig in 0.1 ml, all animals died (100%). Among virus infected with a titer of 3.5 Ig control animals, 80% died Conditions for evaluating antiviral activity Infectious virus titer in Ig in 0.1 ml Enzyme Administration Method The time of death of animals, hour Survival rate,% RNase Bi virus + enzyme 25,000 units (0.033 mg) per 1.0 g of rabbit mass 5.5 intraperitoneal 48-50 (36) thirty intramuscular 48-50 (36) 40 3,5 intraperitoneal 48-60 (48) fifty intramuscular 48-60 (48) 60 RNase RV virus + enzyme 100,000 units (0.33 mg) per 1.0 g of rabbit mass 5.5 intraperitoneal 36 (36) 0 intramuscular 36 (36) 0 3,5 intraperitoneal 48 (48) twenty intramuscular 48 (48) thirty

When animals are infected with a virus with a titer of 5.5 Ig in 0.1 ml, the antiviral activity of RNase Bi at a dose of 25,000 units (or 0.033 mg / 1.0 g weight) varies depending on the method of enzyme administration in the range of 30-40% of surviving animals . At the same time, the infectious titer of the pancreatic RNase virus at a dose of 100,000 units (or 0.33 mg / per 1 g of mass) is 4 times higher than the dose of RNase Bi in activity (100,000 units: 25,000 units = 4) and 10 times - in protein ( 0.33 mg: 0.033 mg = 10), when the titer of the infectious virus 5.5 Ig in 0.1 ml, does not lead to the survival of animals.

When animals are infected with a virus with a titer of 3.5 Ig in 0.1 ml of RNase Bi at a dose of 25,000 units (or 0.033 mg / 1.0 g weight) with an intraperitoneal route of administration, 50% of the animals survive. With an infectious titer of 3.5 Ig virus in 0.1 ml of pancreatic RNase, when administered intraperitoneally at a dose of 100,000 units (or 0.33 mg / per 1 g of mass) 4 times the dose of RNase Bi in activity (100,000 units: 25,000 units = 4) and 10 times the protein (0.33 mg: 0.033 mg = 10) with a titer of an infectious virus of 3.5 Ig in 0.1 ml ensures the survival of 20% of the animals. The antiviral efficacy of RNase Bi with the intraperitoneal method of administering the enzyme without taking into account differences in the used doses of enzymes is 2.5 times higher than the antiviral efficacy of RNase RV 2.5 (50%: 20% = 2.5). Given the differences in the doses used, the antiviral effectiveness of RNase Bi with the intraperitoneal route of administration is higher than the antiviral effectiveness of RNase RV 10 times (100,000 units: 25,000 units) × 2.5 = 10, 25 times the amount of enzyme protein (0.33 mg : 0.033 mg) × 2.5 = 25 With intramuscular injection of RNase Bi at a dose of 25,000 units or 0.033 mg / per 1.0 g of mass, 60% of the animals survive, RNase RV used at a dose of 100,000 units or 0.33 mg / per 1 g of mass, provides survival of 30% of animals. Without taking into account the differences in the doses used, the antiviral effectiveness of RNase Bi is higher than the antiviral effectiveness of RNase RV 2.0 times (60%: 30% = 2). Given the differences in the doses used, the antiviral effectiveness of RNase Bi is higher than the antiviral effectiveness of RNase RV in activity by 8 times (100000 units: 25000 units) × 2 = 8, higher in protein by 20 times (0.33 mg: 0.033 mg) × 2 = twenty.

Example 5. Antiviral activity of the inventive RNase Bi and RNase RV (prototype) upon infection of mice with Aujeszky's disease virus (WBA).

Work with mice is performed according to a known technique [5]. Evaluation of antiviral activity in mice is as follows. Ten groups of white outbred mice weighing 18-20 g (in each group of 10 animals) are taken, which are injected subcutaneously with 10 LD ₅₀ (LD ₅₀ is a lethal dose that causes the death of 50% of the animals) of VBA pathogenic for laboratory animals. After infection of the animals of the two control groups, an isotonic sodium chloride solution is administered, one group intramuscularly, the other intraperitoneally.

The four groups of infected animals are administered the inventive RNase Bi. Two of them injected the enzyme at a dose of 17,000 units or 0.02 mg / 1.0 g of the mass of mice, one group intraperitoneally, the second intramuscularly. The remaining two groups of mice are injected with RNase Bi at a dose of 30,000 units or 0.035 mg / 1.0 g of the mass of mice, one group intraperitoneally, and the second intramuscularly.

Four groups of infected animals are injected with pancreatic RNase (prototype). The enzyme is administered to two groups at a dose of 65,000 units or 0.2 mg / 1.0 g of the mass of mice, one group is intraperitoneal, the second is intramuscularly. The remaining two groups are injected with RNase RVase at a dose of 107250 units or 0.33 mg / 1.0 g of the mass of mice, one group intraperitoneally, and the second group intramuscularly

Compared RNases are administered 1.0 hour after infection of animals for 10 days. After the tenth administration of RNase, animals are observed for another 20 days. The antiviral effect of enzymes is judged by animal survival. The results are expressed as a percentage in relation to the total number of animals in the group (table 5).

The antiviral activity of RNase Bi at a dose of 17,000 units or 0.02 mg / 1.0 g of the mass of mice varies depending on the method of enzyme introduced from 10 to 30% of surviving animals. The life expectancy of infected animals that were injected with the enzyme, but which were not included in the group of survivors, varies from 5 to 8 days, depending on the method of administration of the enzyme.

The antiviral activity of pancreatic RNase at a dose of 65,000 units or 0.2 mg / per 1.0 g of the mass of mice is 3.8 times higher in activity than the dose of RNase Bi (65000 units: 17000 units = 3.8), and the protein exceeds 10 times (0.2 mg: 0.02 mg = 10), with the intraperitoneal and intramuscular route of administration does not lead to the survival of animals.

With an intraperitoneal administration of RNase Bi at a dose of 30,000 units or 0.035 mg / per 1 g of the mass of mice, 30% of the animals survive. RNase of the pancreas with intraperitoneal administration of 107250 units or 0.33 mg / per 1 g of mass does not lead to the survival of animals.

With intramuscular injection of RNase Bi at a dose of 30,000 units or 0.035 mg / per 1 g of mass, 50% of infected animals survive, RNase of pancreas (prototype) with intramuscular injection of 107250 units or 0.33 mg / per 1 g of mass does not lead to animal survival .

The used dose of the claimed RNase Bi (30000 units / ml or 0.035 mg / ml) is 3.6 times less than the dose of the prototype RNase RV (107250 units / ml or 0.33 mg / ml) in activity (107250 units: 30,000 units = 3, 6) and 9.4 times less in protein content (0.33 mg: 0.035 mg = 9.4). The antiviral effect of RNase Bi against BWA has a clear dose-response relationship, whereas RNase RV (prototype) in the studied concentrations, significantly exceeding the concentrations of RNase Bi in activity and protein, does not lead to the survival of animals, but increases their lifespan, which indicates about weak antiviral activity of pancreatic RNase.

Thus, data on the antiviral effect of RNase Bi in vivo indicate that RNase Bi has the ability to suppress the multiplication of DNA-containing viruses, such as Aujeszky's disease virus, which is in accordance with in vitro results (table 2).

Example 6. Evaluation of the antiviral activity of RNase Bi and RNase RV (prototype) when infected with the influenza virus of chicken embryos ("in ovo")

Evaluation of the anti-influenza activity of RNases “in ovo” is carried out on 10-day developing chicken embryos [5]. The drug is administered according to a prophylactic regimen, i.e. 1 hour before the inoculation of the virus, and according to the treatment regimen, i.e. 1 hour after inoculation of the virus. Infectious dose of the virus - 100 EID / 0.2 ml (EID - embryonic infectious dose); enzymes are administered at a dose of 150,000 units / embryo (table 6).

The antiviral activity of the compared ribonucleases (of the claimed and the prototype) is evaluated in equal doses by activity, but the dose of pancreatic RNase for protein is 0.5 mg per embryo, and the dose of RNase Bi is 0.2 mg per embryo, i.e. the dose of pancreatic RNase for protein is 2.5 times greater. The antiviral activity of RNase Bi and RNase RV in relation to influenza virus strains A / PR / 8/34, expressed by the protection index (FR), regardless of the treatment regimen for both enzymes, does not significantly differ and is in the range of 67-71.2% of surviving embryos an infectious dose of 100 EIDs (Table 6).

The antiviral effectiveness of RNase Bi and RNase RV in relation to influenza A virus strains (Odessa) 2882/82 in equal doses in terms of activity and administered according to the prophylactic regimen does not significantly differ - FROM 70.8-72%, FROM for the treatment regimen equal to 76.0 -77.7% (table 6). Given the differences in the doses of the introduced enzymes for the enzyme protein, the antiviral effectiveness of RNase Bi is 2.5 times higher than the effectiveness of RNase RV (prototype).

RNase Bi is effective against human type B influenza virus (Table 7).

Table 7 The antiviral activity of the inventive RNase Bi against influenza B virus (B / Leningrad / 369/75) when infected with the "in ovo" chicken embryo virus. The infectious dose of the virus is 1080 EID ₅₀ / 0.2 ml (embryonic infectious doses causing the death of 50% of the embryos). - RNase Bi at a dose of 0.5 mg / embryo
I3 - The protection index is calculated by the formula: FROM = ((Ms-Me) / Ms) × 100%, where
Ms and Me - mortality in the control and experimental groups, respectively A drug Antiviral activity, protection index (I3)% Number of embryos Introduction scheme I3,% RNase Bi thirty Preventative 65.1 29th Medical 67.3

The antiviral activity of RNase Bi against influenza B virus at an infectious dose of 1080 virus EID ₅₀ / 0.2 ml, regardless of the method of treatment, ranges from 65-67%. Data on the effect of pancreatic RNase on infection caused by influenza B virus are absent (not detected).

Example 7. Evaluation of the antiviral activity of RNase Bi and RNase RV (prototype) when infected with the mouse influenza virus

Evaluation of influenza activity on white outbred mice [5] weighing 16-18 g is carried out as follows. The treatment and prophylactic regimen of the drug is administered intramuscularly, i.e. 24 and 1 hour before infection, as well as 24, 48 and 72 hours after intranasal infection of animals under light ether anesthesia [5]. A single dose of RNase is 750,000 units / kg. Infectious dose of the virus 5 LD ₅₀ / 0,05 ml (table 8).

Table 8 The anti-influenza activity of the claimed RNase Bi and RNase RV (prototype) upon infection of white mice with influenza virus A / PR / 8/34. FM - The protection index is calculated by the formula: FM = ((Ms-Me) / Ms) × 100%, where Ms and Me are the mortality in the control and experimental groups, respectively A drug * Method of drug administration Single dose in mg / kg The number of mice in the group The effectiveness of the treatment of animals on the 8th day Mortality,% FROM% Control (placebo) Intramuscularly - eighteen 77.6 - RNase Bi Intramuscularly 750,000 units (1.0 mg / kg) 16 25.0 67.7 RNase RV Intramuscularly 750,000 units (2.5 mg / kg) 22 81.8 Not active

When treated with a RNase Bi therapeutic treatment scheme for mice infected with 5 LD ₅₀ of the A / PR / 8/34 virus, with an enzyme dose of 750,000 units or 1.0 mg / kg for 5 days, the protection index is 67.7%. RNase RV at a dose of 750,000 units or 2.5 mg / kg, used for treatment according to the same scheme, did not have anti-influenza activity.

The above examples 1, 2, 3, 4, 5, 6, 7 of the manifestation of the antiviral effectiveness of ribonucleases clearly indicate that RNase Bi, taking into account the difference in the dosages of enzymes used and the effectiveness of suppressing the multiplication of viruses, is more active than the prototype pancreatic RNase (pancreatic enzyme) .

For example, the effectiveness of suppressing the multiplication of foot and mouth disease virus (VJ) by RNase Bi (claimed) compared to RNase RV (prototype), taking into account differences in the final titers of the virus and differences in doses of the claimed enzyme and prototype, is 9 times higher in activity and 21 times in protein (Example 1, Table 1).

The effectiveness of suppressing the reproduction of BWA by RNase Bi (the claimed enzyme) compared with RNase RV (prototype), taking into account the differences in the final titers of the virus and the differences in the doses of the claimed enzyme and prototype, is 12 times higher in activity and 28 times higher in protein (Example 2, Table 2).

The inventive RNase Bi, taking into account differences in the number of surviving animals and differences in the doses of enzymes used, is more effective than RNase RVase (prototype) prevents the death of foot-and-mouth disease infected mice by 15 times in activity and 37.5 times in enzyme protein (Example 3, Table 3).

The inventive RNase Bi of a different (with respect to the Bacillus intermedius 7P strain) strain, taking into account differences in the number of surviving animals and differences in the used doses of enzymes, is more effective than the antiviral effectiveness of pancreatic RNase (prototype) prevents the death of foot-and-mouth disease-infected rabbits by 8 times, according to protein 20 times (Example 4, Table 4).

The inventive RNase Bi is more effective than RNase RV in relation to the DNA-containing virus of Aujeszky's disease (WBA). RNase Bi, a 3.6-fold lower dose compared to the prototype RNase RV in activity and 9.4-fold lower in protein content, has antiviral efficacy against BW and has a clear dose-response relationship. RNase RV (prototype) in the studied concentrations, significantly higher than the concentration of RNase Bi in activity and protein, does not lead to the survival of animals, increasing only their lifespan (Example 5, Table 5). This indicates a weak antiviral activity of pancreatic RNase against DNA-containing virus.

The anti-influenza efficacy of RNase Bi and RNase of the pancreas “in ovo” (when infected with chicken embryo influenza A virus), evaluated in equal doses by activity, does not significantly differ. However, the dose of the enzyme protein RNase Bi introduced into the embryos is two and a half times lower. The anti-influenza effectiveness of Bi RNase, estimated by the amount of enzyme protein introduced into embryos, is 2.5 times higher than the anti-viral effectiveness of pancreatic RNase (Example 6, Table 6).

RNase Bi inhibits the reproduction of not only type A virus, but also type B virus. There are no data on the ability of pancreatic RNase to suppress the reproduction of type B virus (Example 6, Table 7).

In the treatment with RNase Bi mice infected with influenza A virus, the animal protection index was 67.7% (with a mortality rate of 25%). Pancreatic pancreatic RNase at the same dose in activity (2.5 times greater in enzyme protein) in the defense index is not active (mortality 81.8%) (Example 7, Table 8).

Similar results of the suppression of virus reproduction were obtained using RNase of another strain of Bacillus intermedius, structurally identical to the RNase of the original strain of Bacillus intermedius 7P described in the above examples (example 4).

The examples of the alleged invention show its usefulness in medicine and veterinary medicine. The advantages of the claimed RNase (RNase Bacillus intermedius) no compared with the prototype (pancreatic RNase RV) are as follows.

1. Inhibition of viral reproduction by Bacillus intermedius RNase is achieved using lower doses of the enzyme (for mammals in vivo, 12.0-20.0 times lower in enzymatic activity, and 20.0-37.5 times lower in enzyme protein), introduced into the infected body. This allows you to reduce the amount of enzyme that must be introduced into the body to achieve the desired effect of suppressing the reproduction of viruses. A decrease in the amount of enzyme introduced into the body reduces the risk of immune complications caused by a foreign protein introduced into the body; in addition, the risk of stimulation by the enzyme of the allergenic properties of other substances entering the body against the background of RNase administration is accordingly reduced. These factors increase the effectiveness and efficiency of the treatment process.

2. The production of bacterial RNase Bacillus intermedius can function stably, regardless of the presence, location, season and workload of meat processing plants, suppliers of raw materials for the production of the prototype pancreatic RNase.

3. The production of the inventive RNase Bacillus intermedius can be placed regardless of the location of the meat processing plants - suppliers of raw materials for the prototype.

4. The use of Rnase Bacillus intermedius to suppress the multiplication of viruses eliminates the deficiency of RNase as a drug, meets the needs of medicine and veterinary medicine in RNase - a tool for the treatment of viral infections.

The present invention meets the criteria of novelty, since the claimed method of using Rnase Bacillus intermedius to suppress the multiplication of viruses is implemented at significantly lower doses compared to the prototype and suppresses the multiplication of viruses significantly more effective than analogues and prototype.

The method of using Bacillus intermedius RNase to inhibit the reproduction of viruses has an inventive step, since no methods have been identified for using other RNases to suppress virus reproduction with the same efficiency as the claimed method of using Bacillus intermedius RNase. The inventive method allows much more effective (from 2 to 37 times compared with analogues and prototype) to suppress the multiplication of viruses in infected organisms of mammals and other warm-blooded animals.

The inventive method allows the use of a previously unknown means for suppressing the multiplication of both RNA- and DNA-containing viruses in infected mammals and warm-blooded animals. The identification of such a means of suppressing the reproduction of viruses in medicine and veterinary medicine expands the list of drugs (compared with the list of known drugs) for the prevention and control of infectious diseases of viral origin.

The inventive method is safe, the components used in the isolation of the bacterial ribonuclease Bacillus intermedius are absolutely harmless to humans. The method allows to successfully treat mammals and warm-blooded animals from a wide range of diseases of a viral nature - biological objects not available for the prototype and analogues, that is, the claimed invention has an extended (compared with the prototype) scope. The results of the application of the proposed method confirm its novelty. The inventive method is feasible using standard technical devices and equipment in the industrial production of medicinal products, in the activities of health organizations, livestock. This corresponds to the criterion of "industrial applicability" presented to the inventions.

The proposed method is useful and necessary for use by specialists in the field of medicine and veterinary medicine, for example for the prevention and treatment of viral diseases of humans and animals. Examples of the application of the invention show its usefulness. The application of the proposed method expands the list of drugs used for medicinal purposes, for example, in the fight against acute viral infections.

The present invention satisfies the criteria of novelty, since when determining the level of technology, no means have been found that have characteristics that are identical (that is, matching the functions performed by them and the form in which these signs are performed) to all the signs listed in the claims, including the purpose of the application.

The inventive method of using ribonuclease Bacillus intermedius has an inventive step, since no technical solutions have been identified that have features that match the distinguishing features of this invention, and the influence of the distinctive features on the specified technical result is not known.

The claimed technical solution can be implemented in the industrial production of medicines, in the activities of organizations of the pharmaceutical industry, healthcare, livestock through the use of well-known standard technical devices and equipment. This meets the criterion of "industrial applicability" presented to the invention.

Information sources

1. Detinenko L. D., Klimenko V. P., Podgorny V. F. and others. Means "Endoglukin" for the prevention and treatment of viral diseases of bees and stimulate the development of bee colonies. Description of the invention to patent RU 2038776, IPC 6 A01K 51/00. Priority from 01/22/1993. Published on July 9th, 1995.

2. Leonova N.S. The use of bacterial endonuclease for the recovery of viruses from potatoes. Leonova N.S., Salganik R.I. // Siberian Bulletin of Agricultural Science, 1991, No. 5. - S.25-28.

3. Batalina T.A., Likhoshvay E.V., Penzikova G.A. Investigation of the effect of Actynomyces rimosus ribonuclease on the reproduction of certain viruses // Antibiotics, 1977, v. 22, No. 1, 25-28.

4. Leshchinskaya I. B. Nucleodepolymerase saprophytic bacteria. - Kazan: Kazan University Press, 1975, 180 p.

5. Starke G. Practical Virology. - M.: Kolos, 1970 .-- S, 126-128.

6. Karyshva A.F., Syurin V.N. Guide to Practical Virology (Reference Guide). - Chisinau: Publishing House "Shtinitsa", 1980. - 212 p.

Claims (2)

1. The method of using bacterial ribonuclease to suppress the reproduction of viruses, which consists in using the bacterial enzyme Bacillus intermedius 7P RNase to treat acute viral diseases of mammals and warm-blooded animals to suppress the multiplication of RNA and DNA-containing viruses. 2. The method according to claim 1, characterized in that they use a structurally identical RNase of the original Bacillus intermedius 7P strain to the ribonuclease of other Bacillus intermedius strains — selected, cloned, and engineering strains.