Antiviral agent

 

(57) Abstract:

The invention relates to medicine and pharmacology, and relates to means for the treatment of herpetic infections. Proposed new antiviral ointment composition based component of national drug interferon inducer complex sodium salts of a single- , double-stranded ribonucleic acids from Saccharomyces cerevisiae. The composition can be applied with greater efficiency for dermatological treatment of lesions caused by the herpes virus has a prolonged action and harmless to the body. 1 Fig., table 4.

The invention relates to the field of medicine and pharmacology, and relates to means for the treatment of herpes infections.

Interest in the study of infections caused by the human herpes simplex virus, remains constant over the last thirty years. This is due to a significant increase in the incidence of genital herpes, ophthalmic herpes, neuralgic herpes and so on, the Number of patients in our country, patients with various forms of herpetic disease every year 2.5 million people.

In this regard, the development of highly effective anti-herpes drugs awn, but it can be simple to manufacture and use. Promising in this direction are drugs that do not require parenteral or injected, i.e., drugs topical use, in particular ointment.

For some infections it is shown that efficient is the application method of application of chemotherapeutic agents [1] or inducers of interferon, is the influence of the drug at the site of viral replication or external manifestations of the disease [2, 3].

Known drug Acyclovir" company Glaxo Wellcome (UK) and its derivatives [8] for the treatment of herpes and other viral infections, which is an acyclic nucleoside analogue of guanine, obtained by synthesis. Ointment form contains 3% of acyclovir and is used to treat Herpes simplex genital [4]. When this ointment is applied on the affected area 5 times a day for 3 days. However, even such intensive use of ointments at an early stage of infection is not efficient enough, as it is accompanied by the accumulation of the virus in the pustules and the formation of erosions in the affected areas. Ointment Acyclovir", with its ability to arrest the acute phase of the disease, does not prevent the development ü "Laifan" (made in Latvia) - high-molecular inducer of interferon natural origin, representing double-stranded RNA phage f2with a molecular weight above 200,000 in [5 prototype].

"Laifan" exerts a protective action against various viral infections, possesses immunomodulatory and antibacterial effect. Used as a therapeutic and preventive agent for arboviral and rhabdovirus infections and therapeutic - when herpes diseases.

The ointment contains 0.05% of lariana. It is applied on the affected area 3-4 times a day. The level of interferon in the serum reaches maximum values in 6-8 hours But within a day the induction of interferon is significantly reduced, which prolongs the course of treatment for 10-14 days. Another drawback ointment "Laifan" is that an active basis it makes phage nucleic acid that represents a potential danger for a man in connection with the recent discovery of massive contamination by phage virus vaccines.

An object of the invention is the creation of new antiviral agents based on ds as inducer of interferon, which can be used with greater effeciencies and harmless to the human body.

The proposed antiviral agent in the form of a topical composition contains 0.15 to 0.5 wt.% complex sodium salts of single - and double-stranded ribonucleic acids from Saccharomyces cerevisiae (complex ds RNA) enclosed in the aqueous phase of emulsion bases of the following composition: liquid paraffin 20% tween - 80 - 17%, emulsifier T-2 (13%), chlorhexidine digluconate - 0.04%, and water to 100%.

The essence of the invention lies in the fact that the proposed antiviral agent - ointment containing as a drug complex of sodium salts of single - and double-stranded ribonucleic acid of killer strains of Saccharomyces cerevisiae (complex ds RNA). This complex is a biologically active part of the drug "Radostin", which is known as an inducer of interferon, which has high efficiency, low toxicity and good tolerability (6). Radostin is a sterile lyophilized solution of the above complex of sodium salts ds in 0.9% isotonic NaCl.

In 1993 the decision of the Presidium of the study of the Pharmacological Committee of the Russian Federation recommended the adoption of the first national inducer of interferon Radostina as protivovirusnoj the th potential of Radostina, it can be used as the basis for creating the ointment form of antiviral agents.

The ointment is prepared as follows: vaseline oil, tween-80 and emulsifier T-2 are mixed in the desired weight amounts and heated to 75oC until complete melting of the mixture, distilled water, heated to 75oC, slowly pour in the molten mass under stirring, reduce the temperature to 40oC and enter the complex ds RNA dissolved in parts of distilled water required for the recipe, and chlorhexidine digluconate. Mixing leads to the formation of stable emulsions.

When conducting clinical trials proposed ointment shown high antiherpetic activity, tolerability and ability to significantly reduce the frequency of relapses.

On the model of genital herpes in Guinea pigs demonstrated a pronounced effect patent-ointment with 3-fold application (see tab. 1), which is considerably lower than the recommended schemes other ointments.

Comparison of the effectiveness of the drug "Acyclovir" and the claimed ointment form shows that previous use of ointments containing comovirus is accompanied by accumulation of the virus in the pustules in the first 3 days. Compared with acyclovir multiplicity of application of the claimed ointment is smaller and amounts to 3 applications (acyclovir-5).

In connection with the obtained results it can be assumed that the drug induces the production of endogenous interferon, which in turn prevents both the multiplication of herpes simplex virus type 2, and reduces inflammation in this disease. The claimed ointment form has an anti-herpes action that is shown on the model of the herpes genitale (Guinea pigs).

In experiments on Guinea pigs was assessed level of interferon in various periods of time after application of the ointment of the inductor. When a single application of ointment is found somewhat higher levels of interferon after 24 h (see table. 2). Further (maximum) increase recorded after 3 days.

Comparison of the interferon-inducing activity of patentable ointments and known drug "Laifan" (made in Latvia) shows that the level of interferon in the serum in the application of lariana reaches maximum values after 6-8 h and 24 h approaches trace amounts, whereas the higher titer of interferon after application of ointments containing complex is it. This fact clearly indicates prolonged therapeutic effect of a new antiviral agents.

The study of the biological effects of the claimed ointment showed that when a single local application in mice increased phagocytic activity of macrophages (see tab. 3), and in Guinea pigs induced production of serum interferon (PL. 2). Preserving the basic properties of the interferon inducer in ointment form shows correctly selected the composition of the filler.

The ointment should be stored at 4oC during the year. The resulting composition has the following physicochemical characteristics:

Authenticity - the integrity of the double-stranded RNA after cooking ointment detected by electrophoresis in 1% agarose gel: in lanes containing samples ointments, there are two bands corresponding to L and M fragments ds in the reference sample of Radostina (Fig. 1)).

PH value of 5.5 to 6.5

Phagocytophilia activity,% not less than 125, P 0.05

Microbiological purity, CFU/g not more than 100

If kept in the course of the year figures have not changed and meet the requirements of the global Fund of the USSR XI.

The invention is illustrated in the following certmgr/ml; track 2-4 - ointment in 2 -, 4-and 8 - fold dilution.

The invention disclosed in the following examples:

Example 1. Preparation of antiviral agents for external use.

The ointment is prepared in a heat resistant glass with a volume of 600 ml. On the scales weigh 100 g of paraffin oil, 65 g of emulsifier T-2 and 85 g of tween-80. The glass is placed in a water bath heated to a temperature of 75oC, and incubated for 25-30 min until complete melting of the mixture.

245 g of distilled water is heated to a temperature of 75oC and slowly pour in the melted mixture with constant stirring. Cooled to a temperature of 40oC with constant stirring and introducing 1 g of chlorhexidine digluconate.

0.75 g of the complex of sodium salts of single - and double-stranded ribonucleic acids in Saccharomyces cerevisiae dissolved in 5 ml of distilled water and injected into the mixture at a temperature of 37oC; continuing to mix, cooled to a temperature of 25oC.

Ointment Packed in bottles of dark glass 5 g and cover with plastic lids.

Example 2. The definition of authenticity antiviral agents.

The definition of authenticity ointment form complex ds osushestvila in 1% agarose gel. For this sample solution ointment (1 g ointment in 3 ml of electrophoresis buffer pH 8,05) diluted in 2, 4, 8 times the buffer solution and put in 20 µl of samples obtained in a mixture with 5 ál of bromophenol blue and sucrose on the tube with 1% agarose gel. As a control, use the sample of Radostina. Electrophoresis spend 1-1 .5 hours at a current of 10 mA per tube.

After electrophoresis the gel is stained in a solution bromistogo ethidium within 15 min, washed with distilled water and viewed under ultraviolet light using chromoscopy.

The results of electrophoresis is shown in Fig. 1. On the tracks (2, 3, 4) containing samples ointments, clearly visible are two strips of double-stranded RNA corresponding to L and M-slices ds in the reference sample of Radostina (lane 1).

Example 3. therapeutic effect of antiviral ointment.

The study of specific activity ointment form complex ds carried out on the model of genital herpes (herpes type 2 strain MS from GKV RAMS) male Guinea pigs. Before use in the experiment viral strain passedout twice on cell culture Vero E6. The titer of the virus is 106JRC.

In the experiment take 48 animals, which are divided into 6 groups. Animal who's receives therapy in the form of topical applications through 1, 24 and 48 h after infection. The second group of animals receives the drug "Radostin", pre-dissolved in 3% glucose, in the form of applications on the place of infection in the same period. The third group of animals receives the same pattern comparator drug - vaseline. The fourth group is the positive control (infected animals and do not receive any treatment), and the fifth group is the negative control, i.e., healthy animals, which compares the intensity of herpetic changes. therapeutic effect is estimated by the difference between points in the 4th - 1st experimental groups during the period of maximum manifestation of the disease (3-4 day after infection).

The results presented in table. 1, indicate that in the groups of animals that received no treatment topical form of the drug (groups 2-4), developed the clinical picture of herpes infection in the first two days, there was a slight swelling and redness at the site of infection for 72 h identifies some pustular phenomena, which are then (96-120 hours) accompanied by severe hyperemia and occurrence of erosion on the site of the pustules. Animals 3-4 groups at 96 h after infection of pustular elements or erosions was isolated herpes virus type 2 102

For the animals of the first group observation lasted 60 days. These animals are marked effect edema external manifestations of herpes infections after applying the ointment form. Throughout the observation period is not marked by relapses, discharge of the virus in the place of the infection, and blood.

Thus, ointment form complex ds has a pronounced anti-herpes activity. Antiviral effect of the drug due to its form and method of application. In the control groups, no vaseline, no local applique dissolved form or injection does not provide antiviral effect.

Example 4. Evaluation of the interferon-inducing activity ointment complex ds RNA.

Studied For this form 4 groups of 8 animals each. Animals of the first group on the outer surface of the penis is applied in the form of application of the ointment. The second group - Radostin dissolved in 3% glucose. The third group - the vaseline. All preparations applied once. The fourth group is intact control. In animals after 1, 3, 5 and 7 days take blood to determine the titers of interferon. Production of serum interferon in animals determine the cytopathic test cell culture L-68, using as a test strain of the virus EMC. The results of the experiment are presented in table 2.

The analysis of the presented data shows that the level of baseline serum interferon in all experimental groups the same and is 2-4 IU/ml After 1, 3, 5 and 7 days in animals 2-4 groups were observed production of serum interferon. On the contrary, in the first group after a single application of ointment found some increased level of interferon in 24 hours Further increase recorded after 3 days and 7 days of interferon indices in this group do not differ from the data of the other three groups.

After 1, 3, 5 and 7 days in animals 2-4 groups is not observed production of serum interferon. On the contrary, in the first group after a single FDA is back after 3 days and 7 days of interferon indices in this group do not differ from the data of the other three groups.

Thus, ointment form complex ds RNA in the local application has the ability to induce interferon in Guinea pigs. Characteristically, the latent period of production of serum interferon caused by the ointment, longer than in the case of use of the injectable form of Radostina. If the highest titers of interferon after parenteral application of Radostina register 3-6 h, when greasing applications in 3 days.

Example 5. Determination of biological activity of the ointment form of the complex ds RNA.

It is known that the herpes viruses are able to inhibit the functional activity of macrophages and interferon synthesis, which ultimately affects the inability to eliminate the virus from the body (7). It is obvious that the selection of promising Antiherpes virus effect funds should assess their effect on phagocytes.

The biological activity of the claimed ointment form estimate on phagocytic activity in the monolayer culture of peritoneal macrophages of mice ICR males. On cviewaction the shoulder of the neck of mice cause the ointment of Radostina in the number of 30-32 mg / cm2surface and rubbed in for 10-12 seconds. In the control groups using the Foundation 4, 48 h after rubbing the ointment. Macrophages isolated from the peritoneal cavity of mice by leaching environment Hanks and heparin (5 units act. on mg medium). Cultivation and all subsequent operations are performed in an environment Hanks with 3% bovine serum. Suspension of macrophages (1106in ml) were seeded on cover glass placed in tube diameter 35 mm, Incubation is carried out 1 h at a temperature of 37oC. For the study of phagocytosis monolayer washed from reprecipitate cells and as the object of phagocytosis put 20 μl of the suspension opsonizing sheep red blood cells (EB) rate of 20 erythrocytes per macrophage. A monolayer of macrophages continue to incubate for 45 min, washed, dried, and prepared for microscopic analysis.

Assessment of phagocytosis carried out by counting peritoneal macrophages that engulfed the sheep erythrocytes.

Phagocytic activity (FA) value in % of the total number of macrophages. The significance of differences (P) data obtained in the experiment and the control, assessed using student's criterion for counting 250 cells on the monolayer. The percentage of stimulation determine the relative performance of the experimental groups of animals to control by the formula:

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The results of these exose macrophages in the experience and control of the same (P = 0.05). However, after 24 h the measure of the functional activity of peritoneal macrophages experimental animals increases and exceeds the control 1.5-2 times (P = 0.05). Two days later found a decrease in phagocytic activity of macrophages (P = 0.05), although groups of this activity remains elevated.

Thus, the study shows that patent ointment form possesses a strong ability to activate phagocytosis. This indicates that therapeutic Antiherpes virus effect of the drug ointment.

Example 6. Defining the boundary of the content of the complex ds in the ointment.

Optimal content of complex ds in the ointment is determined in experiments, stimulation of phagocytosis of macrophages. Studies of phagocytosis of macrophages is carried out after rubbing ointments containing complex ds 0,075%, 0,15%, 0,5%, 0,75%.

The results of these studies are presented in table. 4.

Based on the data, the optimum interval content of the complex ds in the ointment - 0,15-0,50%.

Comparison of the effectiveness of the proposed ointment form and acyclovir (Wellcome, UK) shows that the previous use of the claimed drug does not cause erosion in the lower and amounted to 3 applications (compared to 5 in Edition).

Touching another tool - ointment lariana (Latvia), it should be noted that the greatest effect of its application is also achieved with early application of the ointment. However, it is recommended that 3-fold use of the drug in the day and the course of treatment is 10-14 days.

From these data it is obvious that the ointment form of the complex ds not inferior therapeutic effect ointments with a similar spectrum of activity and output abroad. Given that domestic drug Radostin is used in the treatment and other herpetic diseases, one might think that the ointment form of its biologically active component (complex ds) will be as effective in these cases.

Thus, a new antiviral ointment composition based component of national drug interferon inducer "Radostina", which can be used with greater efficiency for dermatological treatment of lesions caused by the herpes virus has a prolonged action and harmless to the body.

Literature

1. Demicheva E. C., Malinovskaya centuries, Chekunova E. Century//proceedings of the Interferon-89".- M - 1989.- C. 136-139.

2. Xeerchlager Y. , P. Wetzler, K. Thiel - D et al.// Antiviral.Res.- 1982.- v. 2.- p. 255-265.

3. Barinskiy I. F. the ova E. B. // Antivirals (Handbook), St. Petersburg - 1993 - UDC 616.988-085.281.8, S. 23-24.

4. Ershov, F. I., N. Chizhov.P., Tselakova E. B. // Antivirals (Handbook), St. Petersburg - 1993 - UDC 616.988-085.281.8, S. 68-70.

6. The application for invention N 93002333/14 from 12.01.93, "interferon Inducer "Radostin", A. 61 K 37/66.

7. Masycheva Century. And., Sazonov, C. S., Morozova E. N. and other Toxic properties of dsRNA virus-like particles of the yeast Saccharomyces cer.// Antibiotics and chibiterasu - 1986, No. 5, S. 374-378.

8. The international application N 9426273, class A 61 K 31/44, publ. 1995

Antiviral agent-based interferon inducer for topical use, characterized in that it further comprises emulsion base, including vaseline oil, tween-80, emulsifier T-2, chlorhexidine digluconate and distilled water, and as inducer of interferon - a complex of sodium salts of single - and double-stranded ribonucleic acids from Saccharomyces cerevisial, and it is enclosed in the aqueous phase is the basis for the following content, wt.%:

Complex sodium salts of single - and double-stranded RNA from Saccharomyces cerevisial - 0,15 - 0,5

Liquid paraffin - 20

Tween-80 - 17

Emulsifier T-2 - 13

Chlorhexidine biglycan

 

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FIELD: medicine.

SUBSTANCE: method involves carrying out hernia removal in intralaminar way. Posterior longitudinal ligament defect is covered with Tacho-Comb plate after having done disk cavity curettage. Subcutaneous fat fragment on feeding pedicle is brought to dorsal surface of radix and dural sac.

EFFECT: enhanced effectiveness of treatment; reduced risk of traumatic complications.

1 dwg

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