RU2286770C2 - Anti-influenza agent - Google Patents

Abstract

FIELD: medicine, pharmaceutical industry.

SUBSTANCE: invention relates to composition for peroral administration based on paracetamole remantadine, ascorbic acid rutine, loratadine and calcium gluconate. Claimed agent contains two different capsules or tablets which after administration provide therapeutic effect. Agent has febrifuge, antiviral, anti-inflammation, anti-allergic, etc. action, stimulates secretion of interferon-alpha and interferon-beta, and makes it possible to reduce time of influenza and viral infection treatment by 2-3 days.

EFFECT: stable pharmaceutical composition of prolonged storage time useful in large scale production.

10 tbl

Description

The invention relates to medicine, is intended for the treatment of influenza by taking an oral composition based on known drugs. The composition is a finished drug (HFS) with a long shelf life while maintaining its biological activity, suitable for industrial production.

Influenza is a viral disease that has a massive nature during the epidemic. Influenza is often accompanied by complications. It is characterized by an acute onset. The incubation period for influenza A is from several hours to 2 days; for influenza B, it lasts up to 3 days (Influenza. Edited by G.I. Karpukhin, Lenizdat, "Medicine", 1986).

During the flu, the patient suffers from high temperature, headache, fever, general intoxication of the body occurs, the mucous membrane of the respiratory tract, the nervous and cardiovascular systems are affected. In patients, the appearance of muscle and joint pain, general weakness and disturbance on the part of the blood (leukopenia), on the part of the lymphatic system (lymphopenia) are noted.

Influenza treatment is carried out in such a way as to prevent complications, while alleviating the course of the flu (reducing fever, intoxication of the body, headache, etc.), activating the body's fight against viral infection.

For this purpose, antipyretic, anti-inflammatory, antiviral, anti-allergic, general strengthening drugs are used in compositions for treating influenza.

In case of influenza, a patient is prescribed drugs such as acetylsalicylic acid to eliminate the symptomatic manifestations of the disease (Vidal Handbook, Medicines in Russia, 1999, E-5), and other antipyretic and analgesics.

Diphenhydramine is prescribed as antihistamines (M.D. Mashkovsky, Medicines, Moscow, 1987, v. 1, p.309), suprastin M.D. Mashkovsky, Medicines, Moscow, 1987, v. 1, p. 316 ) and similar drugs. They prevent the development of tissue edema caused by histamine, reduce the toxicity of histamine, and reduce the permeability of capillaries.

The best treatment results for influenza are obtained when prescribing complex preparations to patients, i.e. compositions that act on various clinical manifestations of the disease at the same time.

Such compositions are convenient for administration during an illness, as they contain multidirectional medicinal components.

Known for the treatment of influenza composition Coldrex. It consists of paracetamol, phenylephrine, caffeine, terpinghydrate and ascorbic acid (Vidal Handbook. Medications in Russia. 1999, B-307).

The composition of Coldrex includes caffeine, which is a psychostimulant that can disturb sleep, and sleep disturbance is characteristic of the disease itself - the flu.

As a result, this symptom is enhanced when taking Coldrex, which is a disadvantage of this composition. Also unsuccessful is the use of phenylephrine in the composition, which contributes to the violation of hemodynamics in such organs as the lungs and the brain.

The prototype of the invention is a composition consisting of acetylsalicylic acid, ascorbic acid, rutin, diphenhydramine and calcium salts. This mixture of drugs was proposed as a pharmacy prescription (Influenza. Edited by G.I. Karpukhin. Lenizdat, "Medicine", 1986, p. 285).

Its disadvantage is the low analgesic activity.

In the process of treatment with such a composition of drugs, headache, aching joints, and temperature reaction of the body persisted. Long-term use of such a drug due to the presence in the composition of acetylsalicylic acid (more than 50%) can cause an exacerbation of peptic ulcer of the stomach or duodenum.

The aim of the invention is the creation of such a composition for the treatment of influenza, which facilitates the course of the disease and reduces its manifestations (depression of consciousness, headache, drawing pains in the extremities, loss of taste and smell, slowing the pulse, irritation in the trachea and larynx, etc. ), and also has antiviral activity, antitoxic effect.

The composition should be suitable for industrial production and have stability for a long time.

The essence of the invention is the combined use of paracetamol and remantadine in the composition and combinations in leform formulations such as paracetamol, remantadine, ascorbic acid, loratadine, rutin, calcium gluconate. Due to the presence of paracetamol in the composition, it was possible to reduce a single dose of remantadine for adults from 100 mg to 50 mg, obtaining a high drug effect of the composition. To stabilize the composition in industrial production, its components are distributed in 2 capsules or tablets, which are intended for simultaneous administration and which, when taken together, give a high combined therapeutic effect.

The combined drug has antiviral and interferonogenic, antipyretic, anti-inflammatory, analgesic, antihistamine, angioprotective effects.

The choice of components for the composition was based on their well-known pharmacological properties, but the therapeutic effect exceeded expectations.

Apparently, the combination of paracetamol with remantadine enhances the interferonogenic effect, due to which it has become possible in the composition to reduce a single dose of remantadine to 50 mg with a total daily dose of 100-150 mg. The combination of components in the composition makes it possible to alleviate the patient’s condition on the first day of treatment, reducing the duration of the disease by 2-3 days, i.e. a course of treatment for 5 days gives a high percentage of cured patients, no complications of the disease are noted.

The preparations included in the composition were selected taking into account their biological activity. Remantadine (rimantadine) blocks the incorporation of the virus into the host cell, inhibits the release of the viral genome in the cell. The influenza virus has pneumotropic properties, which partly explains the frequency of influenza pneumonia. Due to the presence of the influenza virus in the body, the body's resistance to various secondary infections (pneumococcus, streptococcus, Pfeiffer coli) sharply decreases.

Remantadine has antiviral activity against type A influenza virus (especially A2), reduces the toxic effects caused by type B influenza virus and viruses that cause SARS. It has pronounced interferonogenic properties, increasing the total content of interferons and especially interferon-gamma in the blood.

Therapeutic dose for adults in their pure form is 100 mg 3 times a day or 300 mg once.

Including remantadine in the composition at a dose of 50 mg per single dose, with a total daily dose of 100-150 mg, we obtained a very high therapeutic effect when paracetamol is simultaneously present in the anti-influenza composition.

Paracetamol, like remantadine, is rapidly and fairly completely absorbed in the gastrointestinal tract (GIT). Usually it is prescribed as a painkiller, antipyretic and anti-inflammatory agent. It inhibits the synthesis of prostoglandins (GHGs) and reduces the excitability of the hypothalamic thermoregulation center.

Paracetamol in the compositions with remantadine and used according to the invention the components of the composition of the composition of the composition of the drug previously not used in the drug. The effect of sharing was unexpected.

A single dose of paracetamol when taken in its pure form (outside the composition) is 200-400 mg for adults, 3 times a day. The maximum daily dose is 1500 mg.

In the proposed composition, its daily dose is 700-1100 mg, when taking the composition 2-3 times a day.

It is known that influenza is accompanied by powerful intoxication of the body, complications: inflammation of the accessory cavities, inflammation of the middle ear, catarrhal pneumonia of the aspiration-atelectanic type.

In paracetamol, outside the compositional administration, the anti-inflammatory activity is weak, it is significantly inferior to salicylates, derivatives of pyrazolone. However, in combination with remantadine, with calcium gluconate (100 mg in a single dose) with the antihistamine loratadine (3 mg in a single dose), ascorbic acid (300 mg in a single dose) and rutin (rutoside) (20 mg in a single dose) is anti-inflammatory paracetamol activity increases. Rutin is an angioprotector, it reduces capillary permeability, swelling, inflammation, strengthens the vascular walls, inhibits aggregation and increases the degree of deformation of red blood cells. It is he who gives the composition a greenish-yellowish color. In the claimed composition it was included in the minimum dose known from the literature. Ascorbic acid is involved in redox processes, regulating them. It (vitamin C) regulates carbohydrate metabolism, capillary permeability, blood coagulation, tissue regeneration, and activates immune responses.

In the preparation of HFRS, the presence of ascorbic acid and rutin in the composition does not affect the stability of the composition containing loratadine, remantadine and calcium gluconate, but due to the chemical structure of paracetamol (N - (4-hydroxyphenyl) acetamide, or para-acetaminophenol), mutual the effect of the components, the conversion of paracetamol and other components of the composition into other chemical compounds and, as a result, the composition loses its biological (antiviral, anti-inflammatory, etc.) activity.

Chemical reactions between the components, when all 6 ingredients are in one capsule / tablet, during the storage process violate the overall composition of the leform, i.e. FPP is losing stability. Because of this, the composition was divided into 2 parts according to chemical compatibility, i.e. SFS is 2 capsules or tablets.

The characteristics of the remaining initial components of the composition - loratadine: a pronounced antihistamine effect, prevents the development of tissue edema associated with the release of histamine - and calcium gluconate: prevents the development of increased vascular permeability and fragility, causing hemorrhagic processes in influenza and acute respiratory viral infection (ARVI), and also restores capillary circulation, has an anti-allergic effect. Calcium gluconate is included in the composition at a dose of 100 mg per single dose, which is 10 times lower than the minimum dose per dose in pure form.

Combining drugs, strengthened their positive effect and lowered the negative. As a result of studying the properties of the composition, the quantitative ratios of the components, the optimal ratio of ingredients was selected (in a weight ratio):

Paracetamol 360 mg Remantadine 50 mg Vitamin C 300 mg Routine 20 mg Loratadine 3 mg Calcium gluconate 100 mg

The effect of the proposed composition (in the experiments indicated by PP) was tested in mice and rats. The acute and subacute toxicity, antiviral and interferonogenic effects of the composition proposed on application were evaluated. The acute toxicity of PP in mice was 1100 mg / kg with intraperitoneal administration. A study of subacute (within 15 days) toxicity of the drug PP at a dose of 480 mg / kg (10-50 times the therapeutic dose) compared with the control when administered orally showed the safety of this drug.

The safety of the composition is confirmed by studies of the general condition of animals, their behavior, the blood system, cardiovascular, excretory and digestive systems, post-mortem morphological studies of internal organs.

A double-blind comparative placebo-controlled experimental study of the anti-influenza activity of the composition (PP) showed that it has a pronounced protective treatment and prophylactic anti-influenza effect, which was detected in a model of lethal influenza infection in white mice.

This effect is manifested in all the studied parameters: the index of protection, the duration of the incubation period, as well as the macroscopic manifestations of a typical post-influenza chronic bronchopulmonary process in mice.

The mechanism of antiviral activity of the composition in vivo is due to the induction of serum interferon.

The introduction of PP at a dose of 100 mg / kg stimulated the production of both alpha and gamma interferon, while the total titers reached 73.6-120.6 units / ml by the fifth day. Such indicators of the production of serum interferon upon oral administration of the drug indicates its potential use as an inducer of interferon in the complex treatment of viral diseases.

Thanks to this, the use of the composition in the treatment of influenza, acute respiratory viral infections (ARVI), and febrile conditions involving damage to the bronchopulmonary tree is justified.

Based on the fact that the stability of the composition is maintained when it is divided according to the chemical compatibility of the components, a test for patients was carried out, prescribing the composition in the form of HFRS. Patients took two capsules / tablets (complementary) at the same time orally. The daily intake of the composition according to the invention 2-3 times. The duration of treatment is 3-5 days, because improvement in the condition of patients is noted in a shorter period.

The high clinical efficacy of the complex preparation according to the application is presented in table 1.

The proposed HFRS has an effect both as a means of symptomatic therapy and as a means of influencing the reproduction of influenza viruses.

Table 1 The effect of anti-influenza on the duration of the febrile phase of the disease Group (10 people each) Severity of disease Light (days) Medium (days) Severe (days) Group in experience one 2-3 3-5 Control (without treatment) 2-3 3-5 5-7

Clinical trials of the proposed composition for ARVI patients demonstrated the effect of the claimed HFRS on the immune system and on the functional state of blood macrophages (Tables 2, 3).

table 2 The influence of the anti-influenza composition according to the application on the indicators of the immune system in patients with SARS Indicator Units Control group Group in experience Disease onset End of illness At the beginning of treatment At the end of treatment Lysozyme Mg / L 8.4 ± 0.4 12.1 ± 0.5 10.4 ± 2.1 11.3 ± 1.4 Complement Food 52.0 ± 2.7 52.5 ± 5.3 47.9 ± 4.6 48.8+ 6.2 IgA Mg / L 1b3 ± 13 172 ± 28 155 ± 23 174 + 21 * IgM Mg / L 135 ± 8 134 ± 8 120 ± 12 154 + 17 * IgG Mg / L 194 ± 8 200 ± 15 189 ± 14 205 + 7 * Table 3 The effect of anti-influenza drugs on the functional state of blood macrophages Group of patients The percentage of phagocytic mononuclear cells The percentage of mononuclear phagocytes containing viral inclusions Before treatment After treatment Before treatment After treatment Group in experience 8.4 + 3.0 33.5 + 6.2 * 49.7 + 13.2 12.0 + 4.2 control 11.0 + 3.0 20.4 + 7.1 47.1 + 7.5 32.0 + 8.1 Note * - differences with indicators before treatment are significant, p <0.01

For the industrial production of a composition in the form of an OTC drug, it must be stable and meet the requirements of the Pharmacopoeia. The components are grouped by their chemical properties, by compatibility, into two different capsules or tablets.

Capsule (tablet) 1, designated P in tests - contains paracetamol and excipients (for encapsulation or tabletting).

Capsule (tablet) 2, designated P in the tests - contains remantadine, ascorbic acid, calcium gluconate, rutin, loratadine and excipients (for encapsulation or tabletting).

The simultaneous administration of the proposed two different capsules or tablets gives a therapeutic effect due to the preservation of the composition of the claimed medicinal product. The stability of the composition remains for a long time during storage due to the separate storage of chemically incompatible components.

A binary preparation is prepared by mixing dry active (active) and auxiliary components.

The quantitative composition of each capsule: capsule 1 (P): the average weight of the contents of one capsule is 380 mg, with 360 mg of paracetamol and excipients in it - 20 mg. As auxiliary substances use:

= starch (potato or gelatinized starch) 9.0 mg

= milk sugar (lactose) 4.2 mg

= magnesium stearate 3.8 mg,

silicon dioxide colloidal 3.0 mg.

During the manufacturing process, deviations in the mass of the contents of the capsule are ± 10%, i.e. the mass of the contents is 0.342 g - 0.418 g.

Capsule 2 (P): the mass of the contents of one capsule averages 480 mg, when it contains active ingredients and excipients: the active ingredients 473 mg, excipients 7 mg.

The active components of the composition in this capsule:

Remantadine (rimantadine) 50 mg Vitamin C 300 mg Rutin (Rutoside) 20 mg Loratadine 3 mg Calcium gluconate 100 mg

As auxiliary substances use:

Magnesium stearate 4.8 mg Potato starch) 2.2 mg

During the manufacturing process, deviations in the mass of the contents of the capsule are ± 10%, i.e. the mass of the contents is 0.432 g - 0.528 g.

In industrial production, capsules of different fillings are colored with food colors in different colors, which makes them convenient for patients to take.

A similar approach for the release of tablets is two different colors for each composition from the overall composition.

The spread in the mass of the active ingredients included in the composition while maintaining full activity (drug effect) is ± 10%. Thus, the average mass of each of the incoming drugs is equal (in g):

= paracetamol 0.324-0.396

= remantadine 0.045-0.055

= ascorbic acid 0.270-0.330

= routine 0.018-0.022

= loratadine 0.0027-0.0033

calcium gluconate 0.090-0.110

The stability of the composition was checked after 12 months and after 24 months, the contents of each of the capsules (tablets) No. 1 and No. 2 were checked for compliance with the component (qualitative and quantitative) composition in accordance with the requirements of Pharmacopoeia GF XI., Issue 1, 2. Tested according to 10 capsules of each composition 5 batches of compositions. All values of normalized indicators turned out to be normal, i.e. consistent with the claimed composition. In the table. 4 shows average values. Capsules (their contents) retained their original color (compliance with the requirements of the Global Fund XI, issue 2, p.143):

Capsule 1 (P) - a mixture of powder and / or granules of white or white with a creamy or pink tint;

Capsule 2 (P) is a mixture of powder and / or granules of yellow with a greenish tint and white.

Table 4 The results of checking the stability of the contents of the capsules No. 1 and No. 2 constituting the claimed composition. Party number The name of indicators Year of manufacture, 2002. Average weight in g Year of inspection, 2003. Average weight in g Year of inspection, 2004. Average weight in g Color matching one. No. 1 paracetamol 0.362 0.363 0.362 corresponds to Number 2 - vitamin C 0,301 0.302 0.302 - calcium gluconate 0,099 0,096 0,097 - remantadine 0,050 0.051 0,049 corresponds to - routine 0,020 0.019 0.019 - loratadine 0.0029 0.0029 0.0030 2. No. 1 paracetamol 0,357 0.356 0,358 corresponds to Number 2 - vitamin C 0.297 0.299 0.298 corresponds to - calcium gluconate 0,101 0,100 0.102 - remantadine 0.051 0,052 0.051 - routine 0,021 0,020 0,021 - loratadine 0.0031 0.0030 0.0031 3. No. 1 paracetamol 0.342 0.344 0.346 corresponds to Number 2 0.294 0.296 0.296 - vitamin C 0.104 0.104 0.102 corresponds to - calcium gluconate 0,053 0,052 0.051 - remantadine 0.019 0.019 0,020 - routine 0.0028 0.0029 0.0028 - loratadine four No. 1 paracetamol 0.348 0.349 0.350 corresponds to Number 2 - vitamin C 0.310 0,309 0,307 corresponds to - calcium gluconate 0,092 0,094 0,096 - remantadine 0,049 0,049 0,050 - routine 0.019 0,020 0.019 - loratadine 0.0031 0.0031 0.0030 5 No. 1 paracetamol 0.364 0.363 0.364 Number 2 - vitamin C 0.295 0.297 0.297 corresponds to - calcium gluconate 0.103 0.102 0.102 - remantadine 0,052 0,050 0,049 - routine 0,020 0,021 0,021 - loratadine 0.0028 0.0028 0.0031

Checking the stability of the composition, when all the incoming components were combined together in one capsule, showed that violations in the composition are observed already after 12 months visually. When stored at a temperature of + 30 ° C and a relative humidity of 70%, the contents of such a capsule becomes brown, the powder changes shape - sticks together into lumps.

Quantitative determination of ascorbic acid and paracetamol showed their decrease in the composition by 15% after 12 months and by 20-25% after 24 months of storage.

Based on this, the release of the composition in the form of an OTC drug is possible only in the form of two different capsules or tablets having a chemically compatible composition.

Test methods for the composition in experimental studies, confirming the above about its therapeutic effect.

Test samples. In experimental studies, three samples of drugs were studied. The anti-influenza drug (PP) was a yellowish powder, relatively poorly soluble in water. The known anti-influenza drug remantadine and the thiobenzimidazole derivative almide were used as comparison preparations.

Animals. Experimental studies were carried out on white outbred mice weighing 18-22 g, obtained from the Rappolovo nursery RAMS. The mice were divided into 6 groups of forty animals each. In total, 270 animals were used in this work.

Virus. Influenza A virus, strain Aichi / 2/68 (H3N2), obtained from the Museum of the Research Institute of Influenza RAMS and adapted to white mice, was used.

The technique of setting an experimental influenza infection. The tests were carried out in accordance with the methodological guidelines developed at the Institute of Influenza, Russian Academy of Medical Sciences (Ilyenko V.I. Methods for testing and evaluating the antiviral activity of chemical compounds against the influenza virus (guidelines), L., 1977). For this, the test samples were previously dissolved (or resuspended) in a 1% starch solution in boiled water. The drugs were administered in the tested doses, which are listed in Table 5, orally (via a probe) in a volume of 0.2 ml five times according to the following scheme: 24 hours and 1 hour before infection, 24, 48 and 72 hours after infection. Mice in the control group were similarly injected with a 1% starch solution. Mice were infected intranasally with the pathogenic influenza virus A / Aichi / 2/68 under mild ether anesthesia at a dose of 0.1 and 1 LD ₅₀ (lethal dose). The specified scheme provides for the simultaneous testing of the preventive and therapeutic effects of the test compound. The results were taken into account according to the following indicators:

a) a comparison of the mortality of animals in the group of treated and control mice on the 9th day after infection;

b) determination of the duration of the incubation period for the entire group of animals included in the experiment, during the entire observation period (14 days after infection). In this calculation, a conditional assumption is made that the life expectancy of surviving animals is equal to the duration of the experiment (observation period).

c) macroscopic (visual) determination of the intensity of viral damage to the lungs of surviving mice on the 14th day after infection. The assessment was carried out by the presence of typical foci of chronic influenza pneumonia and expressed as a percentage: 0% - no visible lesions, 25% - segmental lesions, 50% - lesions consisting of several merged segments, but not occupying the entire lobe of the lung, 75% - individual lesions lobes, 100% - total lung damage. Statistical processing of the results of macroscopic studies was carried out by comparing confidence intervals of arithmetic mean values at p = 0.05.

The results of the study.

The survival of mice during an experimental influenza infection after oral administration of test samples by them according to the treatment-and-prophylactic scheme is presented in Table 5. As an indicator of antiviral activity when comparing the mortality of experimental and control groups of animals used the protection index (IZ), which was calculated by the following formula:

The results show that the calculated IZ value for PP and almide at an infectious dose of 1 LD ₅₀ is from 52 to 62%, which makes it possible to evaluate these compounds as having sufficiently pronounced antiviral activity. It should be noted that they are inferior to the classic anti-influenza drug remantadine, in which this indicator is 90.9%.

Evaluation of the antiviral effect of the test samples by the duration of the incubation period (Table 6) also confirms the conclusion about the high anti-influenza activity of the studied drugs. In mice that were administered PP and almide, at an infectious dose of 1 LD ₅₀ , the incubation period increased by 1.8-2.2 days compared with the control. However, its greatest increase (by 3.5 days) was observed with the introduction of remantadine.

Table 5 The influence of test drugs on the course of experimental influenza infection in mice Samples of the drug, dose, mg / kg The number of animals in the group The dose of the virus, LD ₅₀ The number of dead animals on the 9th day Protection Index,% Assessment of drug activity ** Almide, 10 mg / kg twenty 0.1 1 / 5.0 * 50,0 ++ 19 one 5 / 26.3 52.3 ++ PP, 10 mg / kg twenty 0.1 1 / 5.0 50,0 ++ twenty one 4 / 20.0 62.9 +++ PP, 100 mg / kg 19 0.1 2 / 10.5 0,0 - twenty one 5 / 25.0 54.5 ++ Remantadine, 50 mg / kg twenty 0.1 0 1000 ++++ twenty one 1 / 5.0 90.9 ++++ Control, placebo twenty 0.1 2 / 10.0 - - twenty one 11 / 55.0 - - Note: * In the numerator - the absolute number of dead animals, in the denominator - this indicator, expressed as a percentage; ** The drug is not active; +, ++, +++, ++++ - the severity of the antiviral effect.

The lungs of surviving mice infected with 0.1 LD _{50 were} examined. The nature of the pathological process indicates that the intensity of the influenza virus-specific lesion of the lung tissue in mice injected with PP and almide was significantly lower compared to the control group of animals. Of the two tested doses of PP, the greatest protective effect for this indicator was manifested at a dose of 10 mg / kg.

Table 6 Calculation of the incubation period for the entire group of animals Drug, dose, mg / kg The dose of the virus, LD ₅₀ Number of mice Of these, fell on the indicated days of the experiment. Total fell The incubation period, days four 5 6 7 8 9 10 eleven 12 13 fourteen Almide, 10 mg / kg 0.1 twenty - - one - - - one - - - - 13,4 one 19 - 2 one - 2 one - one - - - 6 12.3 PP, 10 mg / kg 0.1 twenty - - - - one - - - - - one 13.7 one twenty - - one 2 - one - - - - - four 12.6 PP, 100 mg / kg 0.1 19 - - - - one one - - - one - 3 13.7 one twenty - - 2 2 - one - - - - - 5 12,2 Remantadine, 50 mg / kg 0.1 twenty - - - - - - - - - - - 0 14.0 one twenty - - - - - - - - one - - one 13.9 Control, placebo 0.1 twenty - one - - - one - one - - - 3 13.1 one twenty - one 2 2 3 3 - - - - - eleven 10,4

As a result of a double-blind study, it was found that the intensity of macroscopic lung lesions, in%, on the 14th day of influenza infection is: with the introduction of almide 30%, PP (10 mg / kg) 20%, PP (100 mg / kg) 35% phytopreparation (margali) 63%, remantadine 5%, in the control 55% (p <0.5 compared with the control).

Conclusion

Based on the obtained experimental data, it was found that the tested PP samples have a pronounced protective treatment and prophylactic influenza effect, which was revealed in a model of lethal influenza infection in white mice. This effect was manifested in all three indicators: the index of protection, the duration of the incubation period, as well as the macroscopic manifestations of the typical post-influenza chronic bronchopulmonary process in mice. The in vivo mechanism of the antiviral activity of these compounds may be due to the induction of serum interferon, which was found in mice after administration of these drugs. This is consistent with the literature that drugs that can stimulate the production of interferon gamma in mice provide protection against lethal influenza infection due to the antidepressant effect on the cellular immunity system. Therefore, the drug PP is applicable for the comprehensive prevention and treatment of influenza and other viral infections.

The study of the interferogenic action of PP

In this study, we studied the production of serum alpha and gamma interferon in mice with the introduction of the studied compounds, presumably with interferonogenic activity. The work was carried out on the basis of the laboratory of influenza vaccines of the Research Institute of Influenza, Russian Academy of Medical Sciences, and the laboratory of etiological methods for diagnosing the Research Institute of Children's Infections of the Ministry of Health of the Russian Federation. The study was conducted by a double blind method.

Materials and methods

Test samples. In experimental studies, three samples of drugs were studied. An anti-influenza drug (PP) is a yellowish powder that is poorly soluble in water. As a comparison drug used a derivative of thiobenzimidazole almide. Doses were 10 and 100 mg / kg for PP and 10 mg / kg for almide. Test samples were pre-dissolved (or resuspended) in a 1% starch solution in distilled water. The introduction of drugs was carried out orally (through a probe) in a volume of 0.2 ml.

Animals. Experimental studies were carried out on white outbred mice weighing 18-22 g, obtained from the Rappolovo nursery RAMS. Mice were divided into 5 groups of 25 animals each. In total, 110 animals were used in this work.

Test method for interferons. Interferon was tested according to the method of Finter N.B. (1968) in the modification of O.A. Aksenov using the Sendai indicator virus (mouse variant) and L-929 cell culture. L-929 culture was seeded in polystyrene panels at a concentration of 200 thousand cells / ml, 100 μl per well. A day later, the sera of mice were added to the grown cell layer after administration of the indicated preparations, interferon inducers, at a 1:20 dilution. Each serum was introduced in two types, native and after treatment with the removal of gamma interferon.

The dose of the Sendai test virus was determined in a special experiment by its trituration on the same tissue culture in the range of 1: 25-1: 1600. Testing of the activity of the virus was carried out by the method of quantitative hemadsorption. To do this, a day after making the indicated dilutions of the virus, the tissue layer of the infected cells was washed, dried, and 0.5% suspension of guinea pig erythrocytes was added to the layer. After 30 minutes, washing was carried out from cells not adsorbed onto the formation with a 4-fold phosphate-buffered solution, and dried for 10 minutes in an thermostat at 37 ° C. After that, a layer of infected cells with adsorbed red blood cells was poured with distilled water to lyse red blood cells at 100 μl per well.

Further, the activity of oxidase enzymes was tested in the erythrocyte lysate according to the principle of enzyme immunoassay. Enzymes were determined using an oxidizing system including hydrogen peroxide and orthophenylenediamine. The principle of the method is that the degree of oxidation of this substrate by erythrocyte oxidase enzymes is linearly dependent on the lysis of the erythrocytes adsorbed by the virus, i.e. from Sendai virus activity. Using this modification allows you to detect the virus in color quantitative measure at a wavelength of 495 nm using enzyme-linked immunosorbent analyzers of various types.

The working dose of the test virus was dilution, reflecting the onset of a decrease in the activity of the test virus (Table 7).

Table 7 Calculating the working dose of Sendai test virus Test virus Activity in the enzyme immunoassay Sendai virus (OD - 495 nm) when introduced into the L-929 culture at a dilution of 1: 25-1600 25 fifty one hundred 200 400 800 1600 without virus Sendai 0.366 0.340 0.326 0.350 0.236 0.115 0,060 0.010

Thus, a working dose of the test virus in this study was a 1: 200 dilution. This virus dilution was introduced into the main experiment with test sera for a day at 37 ° C. After a day, the entire experiment was washed in the same way as in determining the working dose of the virus, the enzyme immunoassay was calculated, and the titer of interferon in the studied sera was determined from the calibration curve of mouse interferon activity. Interferons were determined in the blood serum in the native form and after the destruction of gamma-interferon by heating at 56 ° C for 30 minutes and a 2-day treatment with 1 N-hydrochloric acid.

Research results

To study interferon-producing activity, the studied drugs were administered to mice orally through a probe in a volume of 0.2 ml in the following doses: almide 10 mg / kg, PP 10 and 100 mg / kg. The cycle of drug administration was 3 days. After 1, 2, 3, 4, 5 days after the first injection of the drug, blood was taken from 5 mice of each group and serum was obtained. Serum of control animals was obtained before the start of the experiment.

It was established that upon oral administration of almide, interferon began to appear within a day after the first injection - 28.2 units and over the next 5 days its level varied within 30-44 units in 1 ml. Higher interferon values were detected with the introduction of PP. 5 days after the first administration, PP 10 mg / kg stimulated the production of interferon to a concentration of 76.3 units / ml, PP 100 mg / kg - up to 120.6 units / ml (Table 8).

When evaluating the production of alpha interferon (Table 9), it was found that almide 10 mg / kg stimulated the production of alpha interferon for 3 days after oral administration to 40.5 u / ml, in the future its activity dropped to 17.9 u / ml PP had the maximum activity at a dose of 100 mg / kg, when the value of alpha-interferon increased over 5 days from 36.8 to 80.6 units / ml.

Table 8 The concentration of total interferon in the blood of mice after oral administration of interferonogen preparations Drugs, dose, mg / kg Interferon titer in the blood of mice, days after the first injection one 2 3 four 5 Almide, 10 mg / kg 28,2 44.3 40.5 31.1 30,2 PP, 10 mg / kg 17.8 26.5 51,4 55,2 76.3 PP, 100 mg / kg 36.8 46.1 96.0 116.4 120.6

Table 9 The concentration of alpha-interferon in the blood of mice after oral administration of test drugs The drug dose (mg / kg) The titer of alpha-interferon in the blood of mice, days after the first injection one 2 3 four 5 Almide, 10 mg / kg 25.7 36.7 40.5 25.8 17.9 PP, 10 mg / kg 17.8 26.5 38.9 40.6 35.8 PP, 100 mg / kg 36.8 40.8 63.5 79.6 80.6

When calculating the production of gamma interferon (Table 10), it turned out that the minimum activity of this type of interferon was observed in almide. The interferon titer was practically detected after 5 injections of the drug - 12.3 units / ml. With the introduction of PP, gamma-interferon began to appear in the blood of mice after 2-3 days and by the fifth day it was 30.5 units / ml with a dose of 10 mg / kg and another dose of 100 mg / kg - 40.0 units / ml.

Table 10 The concentration of gamma interferon in the blood of mice after oral administration of interferonogen preparations The drug dose (mg / kg) The titer of gamma interferon in the blood of mice, days after the first injection one 2 3 four 5 Almide, 10 mg / kg 2,5 7.6 0 5.3 12.3 PP, 10 mg / kg 0 0 12.5 14.6 30.5 PP, 100 mg / kg 0 15.3 32,5 36.8 40,0

Conclusion

Of the two preparations tested, almide 10 mg / kg stimulated alpha interferon during the first three days after oral administration. At the same time, gamma-interferon was not produced. The best results for interferonogenic activity were obtained after administration of PP. Its introduction at a dose of 100 mg / kg stimulated the production of both alpha and gamma interferon, with total titers reaching 73.6-120.6 units / ml by the fifth day. Such indicators of the production of serum interferon upon oral administration of the drug indicate its potential use as an inducer of interferon in the complex treatment of viral diseases.

Claims (1)

An anti-influenza drug, characterized in that it is made in the form of two separate capsules or tablets, one of which contains paracetamol and auxiliary substances, such as starch, milk sugar, magnesium stearate, colloidal silicon dioxide, and the other - remantadine, ascorbic acid, rutin, loratadine, calcium gluconate and auxiliary substances, such as magnesium stearate, starch in the following ratio of components per dose, mg: capsule or tablet 1: Paracetamol 324-396 Excipients Starch 8.1-9.9 Milk sugar 3.8-4.6 Magnesium stearate 3.4-4.2 Colloidal silicon dioxide 3.0 Capsule or tablet 2 Remantadine 45-55 Vitamin C 270-330 Routine 18-22 Loratadine 2.7-3.3 Calcium gluconate 90-110 Excipients: Magnesium stearate 4.3-5.3 Starch 2.0-2.4