RU2504397C1 - Pharmaceutical composition containing enzyme ribonuclease and glycyrrhizic acid or salts thereof: ammonium or dipotassium or trisodium glycyrrhizinate - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to pharmaceutical industry and represents a composition for treating and preventing viral infections caused by RNA-containing viruses, such as influenza, parainfluenza, parotitis, rubeola viruses, respiratory syncytial virus, rubella virus, rhinovirus, containing an active ingredient in the form of 0.001-0.5 wt % of ribonuclease (RNA-ase) and 0.001-0.5 wt % of glycyrrhizinic acid or a salt thereof: ammonium or dipotassium or trisodium glycyrrhizinate; the carriers are as follows: 0.05-1.0 wt % of a polymer carrier, 0.001-5.0 wt % of β-cyclodextrins or hydrogenised lecitin and acceptable excipients.

EFFECT: invention provides the preserved activity of the enzyme ribonuclease for a longer period of time, the higher stability and potentiated combined antiviral action.

1 cl, 9 ex, 3 tbl

Description

The present invention relates to medicine, namely to a pharmaceutical composition containing an enzyme ribonuclease (RNAse) and glycyrrhizic acid or its salts: ammonium glycyrrhizinate or dipotassium or trisodium, which can be used in medicine for the treatment and prevention of viral infections caused by RNA- containing viruses such as influenza virus, parainfluenza, mumps, measles, respiratory syncytial virus, rubella virus, rhinovirus and others.

There are known topical medicines used for the treatment and prevention of herpes virus infections, containing antiviral components that are not enzymes, for example Zovirax ™ cream (acyclovir) (http://www.vidal.ru/poisk_preparatov/zovirax~27174.htm); capsules and lyophilisate for the preparation of a solution for intravenous administration of Phosphogliv ™ (phospholipids and trisodium salt of glycyrrhizic acid) (http://www.vidal.ru/poisk_preparatov/phosphogliv.htm); Epigen-intim ™ spray (glycyrrhizic acid) (http://www.vidal.ru/poisk_preparatov/epigen-intim.htm); ointments: alpizarin (http://alpizarin.ru/), helepin (http://www.vidal.ru/poisk_preparatov/helepin-d_20830.htm), flacoside (http://ru.wikipedia.org/wiki/% D4% EB% E0% EA% EE% E7% E8% E4) containing plant extracts.

Known drug Interferon alpha-2a, identical to human leukocyte interferon alpha-2 and having immunomodulating, antiviral and antitumor effects. (Reference "Medicines" edited by M. A. Klyuyev, 2001)

La-Cree ™ Lip Balm is a cosmetic product containing licorice extract and bisabolol, used to soften the skin of the lips. (http://vertex.spb.ru/catalog/68/34/la-kri_balyzam_dlya_gub.html)

Known lip balm Uriage Barierderm, containing shea butter, borage oil, unsaponifiable fractions of avocado oil, stearyl glycyrrhetinate, ascorbyl palmitate, tocopherol, used to repair damaged lip skin. (http://www.krason.ru/catalog-bariederm/1740-3620-balzam-dlya-gub.aspx?perf=1)

Known drug Glycyram (ammonium glycyrrrhizinate), used in the form of tablets for the treatment of bronchial asthma and adrenal hypofunction. (http://www.vidal.ru/poisk_preparatov/glycyram-tablets-0.05_27575.htm)

Known combination drug Hexalysis, containing as antimicrobial components - biclotimol and lysozyme, enoxolone isolated from glycyrrhizic acid as an anti-inflammatory component. (http://www.vidal.ru/poisk_preparatov/hexalyse~22640.htm)

Known topical cosmetics used for the prevention of herpes, containing antiviral components that are not enzymes, for example, extracts of medicinal plants and germanium-organic compounds (Antiherpetic preventative lipstick (P16915891585471.100.70) 292472. TU 9124-001-00358210-96 ), glycyrrhizic acid ointment "Herpinat" ointment; ban. 20 ml; No. 77.01.12.915.P.02970.02.00 from the Research Institute of Atherosclerosis of the Russian Academy of Natural Sciences (Russia), INAT-PHARMA (Russia) (http://www.biomedservice.ru/publish/pub16.htm)

A cosmetic enzyme-containing composition is known where the enzyme is selected from the group of proteinases, including trypsin, chymotrypsin, bromelain, papain and ficin, as well as lysozyme, elastase, α-lipase and α-amylase, which is used to treat acne, care for skin and skin cleansing (DE 4305460). The described composition does not contain nuclease enzymes with direct antiviral activity, and therefore cannot be used to prevent viral infections.

Known pharmaceutical preparation "Deoxyribonuclease", produced in the form of lyophilized powder in hermetically sealed vials and ampoules of 0.005; 0.01; 0.025 and 0.05 g. This drug is prescribed for herpetic keratitis, adenoviral conjunctivitis, to reduce viscosity and improve the evacuation of sputum and pus in bronchiectasis and is used topically in the form of a 0.2% solution on isotonic sodium chloride solution (Reference "Medicines" edited by M.A. Klyuyev, 2001).

Known pharmaceutical preparation "Ribonuclease", produced in the form of lyophilized powder in hermetically sealed vials of 0.01 g. This drug is prescribed for diseases of the respiratory tract with difficult to separate sputum, periodontal disease, gingivitis, purulent wounds, trophic ulcers, tick-borne encephalitis, thrombophlebitis. topical application is sprinkled on the wound surface in the amount of 0.025-0.05 g (Reference "Medicines" edited by MA Klyuev, 2001).

Pharmaceutical compositions containing the enzyme RNA-ase and glycyrrhizic acid or its salts: ammonium glycyrrhizinate or dipotassium or trisodium, used in the prevention of viral infections are not described.

Thus, it is an object of the present invention to provide a pharmaceutical composition comprising an RNAase enzyme and glycyrrhizic acid or its salts: ammonium glycirrhizinate or dipotassium or trisodium, which can be used to prevent viral infections caused by RNA-containing viruses, such like influenza virus, parainfluenza, mumps, measles, respiratory syncytial virus, rubella virus, rhinovirus and others.

According to the present invention, there is provided a composition comprising, as an active ingredient, 0.001-0.5 wt.% Ribonuclease (RNAse) 0.001-0.5 wt.% And glycyrrhizic acid or its salts: ammonium or dipotassium or trisodium glycyrrhizinate, 0.001-0, 5 wt.% And acceptable carriers and excipients.

As a preferred embodiment of the claimed invention, there is provided a composition as defined above, further comprising an antimicrobial or antiviral ingredient.

Most preferred is a composition containing RNA-ase and or glycyrrhizic acid or its salts: ammonium or dipotassium or trisodium glycyrrhizinate as the active ingredient and acceptable carriers and excipients, in the following ratio, wt.%:

RNAse 0.001-0.5 glycyrrhizic acid or its salts: ammonium glycyrrhizinate or dipotassium or trisodium 0.001-0.5 sanguirytrin or interferon, or lysozyme 0.001-5.0 β-cyclodextrin or hydrogenated lecithin 0.001-5.0 preservative 0.5-1.0 polymer carrier 0.05-1.0 pH regulator 0.05-1.0 demineralized water 1.00-5.00 hydrophilic base (Polyethylene Oxide 400 and Polyethylene Oxide 1500 in a mass ratio of 70:30) 86.00-98.397

The active ingredients of the claimed pharmaceutical composition are the enzyme: RNAse and glycyrrhizic acid or its salts: ammonium glycyrrhizinate or dipotassium or trisodium. RNAse has the ability to inhibit the development of influenza virus, parainfluenza, mumps, measles, respiratory syncytial virus, rubella virus, rhinovirus and other RNA viruses, without damaging the RNA of the macroorganism's cells (Mashkovsky M.D. Medicines, Volume 2 Moscow, New Wave, 2000, pp. 111-112). Glycyrrhizic acid and its salts are used as an antiviral agent for external and local use. Glycyrrhizic acid is active against DNA and RNA-containing viruses, including various strains of Herpes simplex, Varicella zoster viruses, human papilloma viruses, cytomegaloviruses. The antiviral effect is apparently associated with the induction of the formation of interferon. Interrupts the replication of viruses in the early stages, causes the virion to exit the capsid, thereby preventing its penetration into the cells. This is due to selective dose-dependent inhibition of phosphorylating kinase P. It interacts with the structures of the virus, changing various phases of the viral cycle, which is accompanied by irreversible inactivation of viral particles (which are in a free state outside the cells), blocking the introduction of active viral particles through the cell membrane into the cell, as well as impaired ability viruses to the synthesis of new structural components. Inhibits viruses in concentrations that are non-toxic to normally functioning cells. Virus strains resistant to acyclovir and iodouridine are highly sensitive to glycyrrhizic acid. It also has anti-inflammatory, analgesic and tissue regeneration-enhancing effects both in the early manifestations of viral infection and in ulcerative forms (http://www.vidal.ru/poisk_preparatov/act_1347.htm).

Interferon It is a highly purified sterile protein containing 165 amino acids. Identical to human leukocyte interferon alpha-2a. It has antiviral, antitumor and immunomodulating activity. It is possible that the mechanism of antiviral and antitumor activity is associated with a change in the synthesis of RNA, DNA and proteins. Inhibits virus replication in virus-infected cells. Increases the phagocytic activity of macrophages and enhances the specific cytotoxic effect of lymphocytes on target cells (http://www.vidal.ru/poisk_preparatov/act_549.htm)

Sanguiritrin® has a wide spectrum of antimicrobial activity, acting on gram-positive and gram-negative bacteria (Staphylococcus spp., Streptococcus spp., Enterococcus spp., Shigella spp., Escherichia spp., Salmonella spp., Proteus spp., Acinetobacterpppppppp. , Pseudomonas spp., Serratia spp., Klebsiella spp., Antracoides spp., Cryptococcus spp.), Pathogenic fungi of the genus Microsporum, Trichophyton, Nocardia, Aspergillus, yeast-like fungi of the genus Candida (including polyresistant strains, Actince, and and some pathogenic protozoa (Entamoeba histolytica, Trichomonas vaginalis). The basis of the mechanism of the antimicrobial action of sanguirythrin is the suppression of bacterial nuclease, the violation of the processes of permeability of cell walls, division walls, nucleoid structure. http://www.vidal.ru/poisk_preparatov/sanguiritrin~21839.htm)

Lysozyme (from the Greek. Lysis - dissolution, decay and zyme - sourdough), muramidase, an enzyme of the hydrolase class; destroys the wall of the bacterial cell, resulting in its dissolution (lysis). In the body, it plays the role of a non-specific antibacterial barrier, especially in places of contact with the external environment (tears, saliva, nasal mucosa). Lysozyme was discovered in 1922 by A. Fleming in mucus from the nasal cavity and then found in many tissues and fluids of the human body (cartilage, spleen, white blood cells, tears), in plants (cabbage, turnip, radish, horseradish), in some bacteria and phages and , in the greatest quantity, in egg white. Lysozymes from different sources vary in structure, but are close in action. Egg protein lysozyme is the first enzyme for which a three-dimensional structure has been established by X-ray diffraction analysis and a relationship has been revealed between the structure and mechanism of action (1965); these studies are a significant contribution to the understanding of the mechanisms of enzymatic catalysis in general.

Lysozyme - a protein with a molecular weight of about 14,000; the only polypeptide chain consists of 129 amino acid residues and is folded into a compact globule (30'30'45 Å). The three-dimensional conformation of the polypeptide chain is supported by 4 disulfide (-SS-) bonds. (There are 3 of them in human milk lysozyme, 2 goose egg protein, 2 in goat T ₄ lysozyme; the more disulfide groups, the more stable but less active.) The lysozyme globule consists of two parts separated by a gap; in one part, most amino acids (leucine, iso-leucine, tryptophan, etc.) contain hydrophobic groups; in others, amino acids (lysine, arginine, aspartic acid, etc.) with polar groups predominate. The polarity of the environment affects the ionization of two carboxyl groups (-COOH) located on the surface of the gap of the molecule from different sides. Lysozyme acts on one of the main components of the bacterial wall - a complex polysaccharide consisting of two types of amino sugars. A polysaccharide is adsorbed on a lysozyme molecule in a gap on the border of its hydrophobic and hydrophilic parts in such a way that 6 amino sugar rings bind to the enzyme, and one of the glycosidic bonds connecting them (between 4 and 5 rings) is between the carboxyls. Due to the interactions between the carboxyls of lysozyme and the atoms forming the glycosidic bond, as well as the distortion of the bond angles of the substrate, the bond is activated and broken. This leads to destruction of the bacterial cell membrane.

(Phillips D., Three-dimensional structure of the enzyme molecule, in the collection: Molecules and cells, trans. From English, century 3, M., 1968; Protein chemistry, M., 1969; Bernhard S., Structure and function of enzymes, trans. . from English., M., 1971).

Thus, a complex antiviral effect is carried out: interruption of replication of the virus in the early stages, stimulation of the release of the virion from the capsid, hydrolysis of the viral RNA and induction of the formation of interferon, as well as suppression of secondary microflora and inflammation in the affected area and acceleration of epithelization.

To prepare the composition, the enzyme preparation “Ribonuclease” is used. The preparation was obtained from the pancreas of cattle by extraction from crushed raw materials, followed by purification and freeze drying. Lyophilized dried “Ribonuclease” powder is used for diseases of the respiratory tract with difficult to separate sputum, periodontal disease, gingivitis, purulent wounds, trophic ulcers, tick-borne encephalitis, thrombophlebitis.

RNA-ase in the form of this drug is not used for the treatment and prevention of viral infections due to its difficult absorption through intact skin and a low level of consumer properties, as well as the lack of stability when the moisture content is more than 3% and in solution, with loss of enzyme activity during the day.

Surprisingly, RNAse and hydrogenated lecithin or beta-cyclodextrin can be included in a stable composition while maintaining the activity of the enzyme for a long time, despite the fact that such a composition contains a significant amount of water (up to 5%).

The composition of the present invention is usually made by conventional methods using solid or liquid acceptable carriers or excipients selected from emulsifiers, dispersants, preservatives, flavorings, pH adjusters, polymer carriers and other excipients that are suitable for the preparation of compositions for topical use. The composition of the present invention can be prepared in the form of lipstick, injection, eye drops, suppositories, gel, cream, paste, ointment and the like.

Additional advantages of this composition are increased stability, potentiated complex antiviral effect, ease of use, as well as ease of dosing.

The claimed composition has stability with respect to the activity of the enzymes included in its composition during long-term storage, which is achieved by immobilization of enzymes on carriers contained in the specified composition. As these carriers, any lecithins, cyclodextrins and polymers capable of binding to enzymes to form enzyme-carrier complexes can be used. Examples of such polymeric carriers are gums, polyvinylpyrrolidone, carrageenans, sodium alginate, chitosan polyethylene glycol ether, sodium carboxymethyl cellulose, polyacrylate.

The activity of the claimed composition is due to the activity of the enzyme included in its composition and glycyrrhizic acid or its salts: ammonium glycyrrhizinate or dipotassium or trisodium. It was found that the composition for topical application containing RNA-ase 0.001-0.5 wt.%, Has stability with respect to the activity of the enzyme during its long-term storage, while the activity of this enzyme, prepared in the form of a solution, decreases rapidly. Glycyrrhizic acid or its salts: ammonium or dipotassium or trisodium glycyrrhizinate are stable compounds and do not require stabilization. Therefore, the claimed composition can be used for the effective prevention of RNA-containing viruses, such as influenza virus, parainfluenza, mumps, measles, respiratory syncytial virus, rubella virus, rhinovirus and others.

The present invention is illustrated by the following examples.

EXAMPLE 1

A gel composition based on RNAse and ammonium glycyrrhizinate has the following composition:

RNAse 0.5 Ammonium Glycyrrhizinate 0.5 β-cyclodextrin 5,0 emulsifier 0.5 preservative 1,0 lysozyme 5,0 polymer carrier 1,0 pH regulator 1,0 demineralized water 85.50

The specified composition is prepared according to the following method. RNAse, β-cyclodextrin, ammonium glycyrrhizinate, lysozyme are dissolved in demineralized water, an emulsifier, a preservative, a polymeric carrier, and a pH regulator are added. The resulting gel is stirred, vacuum and homogenized.

EXAMPLE 2

The composition in the form of eye drops based on RNAse and dipotassium glycyrrhizinate has the following composition.

RNAse 0.001 Dipotassium glycyrrhizinate 0.001 Hydrogenated lecithin 0.001 emulsifier 0.5 preservative 1,0 interferon 0.001 polymer carrier 1,0 pH regulator 1,0 demineralized water 96,496

The specified composition is prepared according to the following method. RNAse, dipotassium glycyrrhizinate, interferon are dissolved in demineralized water, an emulsifier, a preservative, a polymeric carrier, hydrogenated lecithin, and a pH regulator are added. The resulting solution is filtered and sterilized.

EXAMPLE 3

The composition in the form of suppositories based on RNAase and trisodium glycyrrhizinate has the following composition:

DNAase 0.5 Trisodium Glycyrrhizinate 0.5 β-cyclodextrin 5,0 emulsifier 0.5 preservative 1,0 sanguirythrin 5,0 polymer carrier 1,0 suppository base 80.5 pH regulator 1,0 demineralized water 5,0

The specified composition is prepared according to the following method. The suppository base is melted at a temperature of 40 ° C, RNAase, β-cyclodextrin, trisodium glycyrrhizinate, sanguirytrin are dissolved in demineralized water, an emulsifier, preservative, a polymeric carrier, and a pH regulator are added. The resulting mass is stirred, homogenized and poured into a mold and cooled.

EXAMPLE 4

The composition in the form of an injection for intravenous and intramuscular administration based on RNAse and ammonium glycyrrhizinate has the following composition.

RNAse 0.001 Ammonium Glycyrrhizinate 0.001 Hydrogenated lecithin 0.001 emulsifier 0.5 preservative 1,0 interferon 0.001 polymer carrier 1,0 pH regulator 1,0 demineralized water 96,496

The specified composition is prepared according to the following method. RNAse, ammonium glycyrrhizinate, interferon are dissolved in demineralized water, an emulsifier, a preservative, a polymer carrier, hydrogenated lecithin, and a pH regulator are added. The resulting solution is filtered and sterilized.

EXAMPLE 5

Assessment of the stability of the enzyme.

Since the main disadvantage of RNAase is its instability in the presence of water from 3%, a comparative assessment was made of the conservation of enzyme activity during storage in the form of a native solution and in the form of compositions from EXAMPLES 1-4.

The activity of RNAse was determined by the following method.

Determination of RNAase activity was carried out according to the Kunitz method. For the unit of activity, RNA-basics take the smallest amount of the enzyme, which when exposed to the substrate under the conditions of the experiment releases such an amount of acid-soluble products, which leads to an increase in optical density at a wavelength of 260 nm per unit. 0.5 ml of substrate (RNA) and 0.3 ml of 0.2 M Tris buffer (pH = 7) are added to the control and two test tubes. In experimental, pre-warmed tubes, add 0.2 ml of the enzyme solution. Samples are thermostated for 30 minutes at 37.5 ° C. Then, 1 ml of a 1N solution of perchloric acid (HClO ₄ ) cooled in an ice bath is added to the samples; 0.2 ml of the enzyme solution is added to the control. The tubes are shaken and kept in an ice bath for 16-25 minutes. The precipitates formed are separated by centrifugation at 4000 rpm for 10 minutes. 0.5 ml of solution was taken from the supernatant, 2.5 ml of water was added and the absorbance was measured at λ = 260 nm.

The results are shown in the following table.

Table 1 Test samples Main ingredients Storage stability one 2 3 Gel number 1 According to Example 1 RNAase activity 90% for 5 years of observation Eye drops No. 2 According to Example 2 RNAse activity 92% for 5 years of observation Suppositories No. 3 According to Example 3 RNAse activity 92% for 5 years of observation Solution for injection No. 4 According to Example 4 RNAse activity 85% for 5 years of observation Native enzyme solution (without carrier) - 5% activity for RNAase within 1 day

As can be seen from the table, the use of polymer carriers and stabilizers in the composition allows to obtain stable compositions while maintaining the activity of the enzyme. during 5 years of observation, due to the creation of a stable conformation of the enzyme with β-cyclodextrin, hydrogenated lecithin and a carrier polymer.

EXAMPLE 6

The study of influenza activity

The study of influenza activity was carried out on white randomly bred male mice weighing 18-20 g. Drugs were administered to mice in doses:

- The composition according to EXAMPLE 4 is 5 ml / kg and 25 ml / kg intraperitoneally (0.1 ml and 0.5 ml per mouse, respectively) for 10 days before infection with the virus.

- The composition according to EXAMPLE 3 is 100 mg / kg and 500 mg / kg rectally for 10 days before infection with the virus.

- Isoprinosine - 500 mg / kg in the form of an aqueous suspension of tablets, calculated on the content of the active component in them for 10 days before infection with the virus. This dose is extrapolated to mice with the recommended daily dose for humans by multiplying by an interspecific transfer coefficient of 10.

In each group, 20 mice were used. The control group of mice (without prophylaxis) received distilled water in a volume of 0.5 ml per mouse (25 ml / kg). On the 11th day after the start of drug administration, mice were infected intranasally under mild ether anesthesia with a strain of influenza virus A / Pr / 8/34 (H0N1) pathogenic for them in a volume of 0.05 ml per mouse. The lethal dose of the virus was determined in a preliminary experiment by titration of the virus in mice, followed by the calculation of LD ₅₀ .

The results of the experiment were evaluated on day 5, recording the mortality of mice in the control and experimental groups.

The evaluation of the test results was carried out on the basis of the following indicators:

- Comparison of the percentage of dead mice in the control and experimental groups;

- determining the protection index of the test drug. To determine the protection index (IZ), the multiplicity or coefficient of protection (CI) of mice was calculated by the preparation.

The calculation of the protection index (IZ) was carried out as follows:

.

.

The test results of the influenza prophylactic effect of drugs in experiments on mice are presented in table 2.

Preventive antiviral activity of drugs in experiments on mice infected with the influenza virus A / Pr / 8/34

table 2 Experience Conditions Dose, mg / kg (ml / kg) Number of infected animals Of these, death was noted. Protection coefficient (KZ) Protection Index (FROM),% amount % Water + Virus flu A / Pr / 8/34 25 twenty 16 80 0 0 (group without Composition according EXAMPLE 2 + 500 twenty 7 35 2.28 56 flu virus Composition, according to EXAMPLE 2 + one hundred twenty 8 40 2.0 fifty flu virus Composition, according to EXAMPLE 4 + 25 twenty eleven 55 1.45 31 flu virus A / Pr / 8/34 Composition, according to EXAMPLE 4 + 5 twenty 12 60 1.3 24 flu virus

Isoprinosine + flu virus 500 twenty 6 thirty 2.7 63 A / Pr / 8/34

According to the data of table 2 in the control group, the virus A / Pr / 8/34 caused 80% death of mice. The administration to animals of the compositions of EXAMPLES 2 and 4 reduced the mortality rate of mice and increased the protection index.

EXAMPLE 7

Evaluation of the effectiveness of the composition against influenza virus

To assess the effectiveness of the composition, a study was conducted of the composition of EXAMPLE 1. The study involved 50 patients aged 18 to 50 years with clinical manifestations of influenza. 25 patients were intranasally administered three times a day for 7 days with the composition of EXAMPLE 1, 25 patients were given symptomatic treatment. In this study, the following clinical effects of the composition from EXAMPLE 1 were identified: acceleration of the relief of fever was noted and prevalence of a critical type of temperature decrease in all age groups (on average in 53% of patients). This effect is explained by the antiviral effect of the composition and is confirmed by a reduction in the duration of detection of viral antigen in smears from the nasal mucosa. The duration of a runny nose and cough is significantly reduced. The marked clinical efficacy of the composition of EXAMPLE 1 was accompanied by a protective effect against subsequent diseases for 6 months. The number of influenza episodes per observed patient turned out to be 3 times less for the treatment compositions of EXAMPLE 1. An important advantage of the treatment with the composition of EXAMPLE 1 was the prophylactic effect on the layering of the secondary bacterial flora: complications were significantly less frequent in the treatment of the composition of EXAMPLE 1 (23, 7%) in contrast to the comparison group (45%) (p <0.05). Evaluation of the transformation of the form of rhinitis into purulent significantly confirmed the indicated effect - 7.9% during therapy with the composition of EXAMPLE 1 and 20% - comparison group.

Evaluation of the antiviral effect of the composition of EXAMPLE 1 was carried out on the basis of PCR diagnostics performed on the 1-2nd and 5-6th days of the disease. In half of the patients receiving the composition from EXAMPLE 1, an etiological agent was not detected on the 5-6th day of the disease, while in the comparison group in a third of patients (p <0.05).

Similar data were obtained in the study of the compositions of EXAMPLES 1-3. Thus, based on the data obtained, we can conclude that antiviral, including and the prophylactic effect of the compositions of EXAMPLES 1-4 against influenza virus.

EXAMPLE 8

Evaluation of the effectiveness of the composition against parainfluenza viruses. endemic mumps, measles and rubella

The composition of EXAMPLE 4 was used in the work. Aliquots of the composition of EXAMPLE 4 were diluted in cell culture medium. Ribavirin at concentrations of 30-300 μg / ml was used as the reference drug. The human parainfluenza virus, endemic mumps virus, measles virus and rubella virus were used in the work. Viruses were cultured in MA-104 cells. A series of ten-fold dilutions from 10 ^-1 to 10 ^-6 on MEM medium was prepared from the starting virus-containing material. Cells were individually infected with human parainfluenza virus, mumps virus, measles virus and rubella virus in the presence of test drugs. The infected cells were cultured at 37 ° C in an atmosphere of 5% CO ₂ in a gas-flow incubator. The cultivation period was 72 hours. Cells were washed 2 times with neutral phosphate buffer, fixed for 10 minutes at + 4 ° C with a solution of 80% acetone, washed for 5 minutes with distilled water and dried in air. The production of viral proteins in fixed cells was evaluated using enzyme-linked immunosorbent assay. Primary type-specific antibodies against parainfluenza, mumps, measles and rubella viruses were dissolved in phosphate buffer supplemented with 5% skimmed milk powder (to block the sites of non-specific binding of antibodies to protein determinants) to a concentration of 5 μg / ml and incubated with fixed cells for 1 hour at 37 ° C (0.1 ml per well). Cells were washed 3 times with 0.1 ml / well of phosphate buffer and incubated with a solution of anti-mouse immunoglobulin labeled with peroxidase at a dilution of 1: 10000 (0.1 ml per well) for 1 hour at 37 ° C. Unbound antibodies were washed 3 times with 0.1 ml / well of phosphate buffer and a color reaction was carried out with a solution of 3,3,5,5-tetramethylbenzidine with the addition of a 0.03% solution of hydrogen peroxide in acetate buffer pH 5.0 (0.1 ml per hole). The reaction was stopped with a solution of 2M sulfuric acid (0.1 ml per well) and the absorbance in the cells was measured at a wavelength of 450 nm on a microplate reader. The reaction was considered positive if the optical density in the cells exceeded the background values for intact cells by half (such a dose of the virus was taken as 50% of the experimental infectious dose, EID ₅₀ ) and more. Virus replication in the presence of drugs was further studied by the manifestation of the cytopathogenic effect of the virus in cell culture. Viruses were cultured in the presence of preparations as described above, the cells were washed 2 times with 5 min phosphate-buffered saline, and the number of living cells was estimated using a microtetrazolium test (MTT), which characterizes the mitochondrial respiration rate of living cells. For this purpose, 100 μl of a solution of (5 mg / ml) 3- (4,5-dimethylthiazolyl-2) 2,5-diphenyltetrazolium bromide in physiological saline was added to the wells of the plates. Cells were incubated at 37 ° C in an atmosphere of 5% CO ₂ for 2 hours and washed for 5 minutes with phosphate-buffered saline. The precipitate was dissolved in 100 μl per well of DMSO, after which the optical density in the wells of the tablets was measured on a multifunction reader at a wavelength of 535 nm. A virus test was considered positive if the optical density in the well was two or more times lower than in control wells without the virus. In both tests, the value for the titer of the virus was taken as the opposite of the decimal logarithm of the highest dilution of the virus, which could cause a positive reaction in half of the infected wells of the plate. The titer was expressed in logarithms of a 50% cytotoxic dose (Ig CTD ₅₀ ). The virus-inhibiting effect of the compounds was judged by the decrease in the titer of the virus in the presence of the drug compared to the corresponding control wells without drugs. Statistical processing of the results (calculation of mean values and standard deviations, as well as calculation of 50% of effective doses using linear regression) was performed using the Microsoft Excel program. Significance of differences was evaluated by student criterion. The differences between groups were considered significant if the parameter "p" did not exceed 0.05. In this study, the antiviral activity of the composition of EXAMPLE 4 against parainfluenza, mumps, measles and rubella viruses was studied using enzyme-linked immunosorbent assay (ELISA).

Data on the effect of the composition of EXAMPLE 4 on the production of specific viral proteins in cell culture are summarized in table 3.

Table 3 Antiviral activity of the composition of EXAMPLE 4 against viruses of para-influenza, mumps, measles and rubella according to the results of enzyme-linked immunosorbent assay Drug concentration Optical density (1000XOD ₄₅₀ ) in wells when diluting the virus Virus titer, Ig EID ₅₀ * 10 ^-2 10 ^-3 10 ^-4 10 ^-5 0 (drug control) The composition of EXAMPLE 4 500 m kg / ml 895 ± 27 854 ± 33 826 ± 6 152 ± 11 136 ± 2 4.0 ± 0.0 The composition of EXAMPLE 4 170 m kg / ml 978 ± 3 866 ± 17 853 ± 10 169 ± 8 137 ± 2 4.3 ± 0.3 The composition of EXAMPLE 4 50 m kg / ml 965 ± 14 862 ± 8 849 ± 24 829 ± 13 156 ± 6 4.7 ± 0.3 Ribavirin 50 m kg / ml 890 ± 16 851 ± 21 754 ± 14 212 ± 9 142 ± 5 4.3 ± 0.3 Virus control 947 ± 6 867 ± 8 836 ± 17 829 ± 14 5.0 ± 0.0 Cell control 137 ± 5 - Note. * - the virus titer is calculated from three parallels on the tablet.

According to the ELISA results, the use of the composition from EXAMPLE 4 led to a 10-fold decrease in viral production in cell culture, while the activity of the composition from EXAMPLE 4 at a maximum concentration was twice the activity of the reference drug ribavirin. Using a regression analysis, a 50% effective dose (EC ₅₀ ) of the composition of EXAMPLE 4 for parainfluenza, mumps, measles and rubella viruses was calculated at 203 μg / ml. From the previously obtained data, a 50% cytotoxic dose of the composition from EXAMPLE 4 is> 1000 μg / ml, which gives a selectivity index> 5. Based on the results obtained, the composition of EXAMPLE 4 should be characterized as a preparation against viruses of the parainfluenza, mumps, measles and rubella. Similar data were obtained in the study of the compositions of EXAMPLES 1-3.

EXAMPLE 9

Evaluation of the effectiveness of the composition against rhinovirus and respiratory syncytial virus

The study of the therapeutic efficacy of the composition of EXAMPLE 3 was carried out in 51 patients with a diagnosis of SARS. The main group consisted of 26 people aged 16 to 64 years, 14 men and 12 women who received the compositions from EXAMPLE 3. Based on PCR diagnostics, 10 people in the main group were diagnosed with respiratory syncytial virus, and 16 people had rhinovirus. Against the background of basic symptomatic therapy, the compositions of EXAMPLE 3 were administered rectally, 1 suppository 3 times a day in the first 2 days of treatment, then 1 suppository 3 times a day for the next 2 days; the control group consisted of 25 ARVI patients aged 15 to 62 years, 11 men and 14 women who received placebo in the same dose against basic therapy.

Laboratory studies included a clinical blood test, a biochemical blood test for urea, creatinine, AlAT, bilirubin, an X-ray of the chest, an ECG. Studies were conducted before and immediately after treatment. The diagnosis of acute respiratory viral infections was confirmed by the method of immunofluorescence express diagnostics and stating RSK and RTGA in dynamics. The diagnosis of acute respiratory viral infections was confirmed by laboratory methods in 88.2% of cases. Clinical examination of patients included daily examination, double thermometry, blood pressure control.

Assessment of therapeutic efficacy was carried out according to the following parameters:

1. Terms of normalization of temperature.

2. The duration of intoxication syndrome.

3. The duration of the catarrhal syndrome.

4. The occurrence of complications in the treatment process.

As a result of the studies, it was shown:

1. A significant reduction in catarrhal syndrome to (4.16 ± 0.33 days) in patients receiving the composition of EXAMPLE 3 from the 1-3rd day of illness, in contrast to the group of patients receiving placebo at the same time of the disease (6.1 ± 0.36 days).

2. The absence of toxicity of the applied composition from EXAMPLE 3 according to the results of laboratory and clinical studies. Good tolerability of the drug was noted, side effects on the background of its appointment were not observed.

The data obtained indicate the therapeutic efficacy of the composition of EXAMPLE 3 against rhinovirus and respiratory syncytial virus.

Similar data were obtained in the study of the compositions of EXAMPLES 1, 2, 4.

Claims (2)

1. Composition for the treatment and prevention of viral infections caused by RNA-containing viruses, such as influenza virus, parainfluenza, mumps, measles, respiratory syncytial virus, rubella virus, rhinovirus, containing 0.001-0.5 wt.% As an active ingredient ribonuclease (RNAase) and 0.001-0.5 wt.% glycyrrhizic acid or its salts: ammonium glycyrrhizinate, or dipotassium, or trisodium, as carriers: 0.05-1.0 wt.% polymer carrier, 0.001-5 , 0 wt.% Β-cyclodextrins or hydrogenated lecithin and acceptable excipients. 2. The composition according to claim 1, additionally containing 0.001-5.0 wt.% Sanguirythrin, or interferon, or lysozyme and acceptable excipients in the following ratio of components, wt.%:
RNAse 0.001-0.5 glycyrrhizic acid or its salts: ammonium glycyrrhizinate, or dipotassium or trisodium 0.001-0.5 sanguirythrin, or interferon, or lysozyme 0.001-5.0 β-cyclodextrin or hydrogenated lecithin 0.001-5.0 preservative 0.5-1.0 polymer carrier 0.05-1.0 pH regulator 0.05-1.0 demineralized water 1.00-5.00 hydrophilic base 86.00-98.397