EA008765B1 - Antiinflammatory preparation - Google Patents

Abstract

The invention relates to medicine and can be used for treating influenza. A composition based on paracetamol, remantadine, ascorbic acid, rutin, loratadine and calcium gluconate is a complex preparation for oral administration.The invention is a medicament dosage form of long term storage and due to the stability of composition is useful for commercial production as a finished medicament.The stability of the composition is achieved by manufacturing the finished medicaments as tablets or capsules, the ingredients of the composition are dispersed in two different tablets or capsules, which provide a medical therapeutic effect when administered simultaneously.When the finished medicament is manufactured as a powder for preparing a solution its stability is provided by including in the powder auxiliary substances, the mass of which exceeds 5 times the mass of the active components, that is active substances.The inventive composition acts upon main influenza symptoms, has antipyretic, antiviral, anti-inflammatory, antiallergenic and tonic action, as well as stipulates production of alpha-interferone and gamma-interferone.The use of the inventive anti-inflammatory preparation for the treatment of influenza and acute respiratory viral infection shortens the treatment by 2-3 days creating no complications.

Description

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

Influenza is a viral disease that has a massive nature during the epidemic, which is often accompanied by complications. It is characterized by an acute onset. The incubation period for influenza A ranges from several hours to 2 days, for influenza B - up to 3 days (Influenza / Ed. By G.I. Karpukhin, Lenizdat, “Medicine”, 1986).

During the flu, the patient suffers from high fever, 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) and on the part of the lymphatic system (lymphopenia) are noted.

Treatment of influenza is carried out in such a way as to prevent complications, while facilitating the course of influenza (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 the case of influenza, a patient is prescribed drugs such as acetylsalicylic acid to eliminate the symptomatic manifestations of the disease (Vidal. Medications in Russia. / Handbook, 1999, E-5), and other antipyretic and painkillers.

Diphenhydramine (M.D. Mashkovsky, Medicines, M., 1987, v. 1, p. 309), suprastin (M.D. Mashkovsky, Medicines, M., t. 1, s. 316) and similar preparations. 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 drug components.

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

The composition of Coldrex includes caffeine, which is a psychostimulant that can disturb sleep, and sleep disturbance is characteristic of the flu itself. 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 and 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, i.e., a symptomatic drug with a drug with antiviral activity, and the simultaneous use in the dosage form of such drugs 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 to 50 mg, obtaining a high drug effect of the composition.

With the desire of modern medicine to work with ready-made medicines, and not with prescriptions for pharmacies, it becomes necessary to create compositions for industrial production that are stable during storage and effective in treating the disease.

The composition according to the invention has been tested in pharmaceutical release forms such as capsules, tablets, water-soluble powders for the preparation of a solution for oral administration according to a treatment regimen, i.e., a drinking medicine.

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To stabilize the composition during its industrial production in the form of capsules or tablets, the components are separated into 2 capsules or 2 tablets, which are intended for simultaneous administration and which, when taken together, give a high combined therapeutic effect.

In the preparation of the powder, stabilization is provided by auxiliary substances, which in the powder by mass are 5 times more than the composition consisting of drugs and providing a therapeutic effect. The main role in stabilization is played by lactose (milk sugar), which is often used for these purposes in pharmaceutical production. The active components are dispersed by weight of auxiliary substances and do not affect each other during storage.

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

The choice of components for the composition was based on 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 acute respiratory viral infection (ARVI). It has pronounced interferonogenic properties, increasing the total content of interferons and especially gamma-interferon in the blood.

The 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) for adults is 200-400 mg, taken 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, such complications as inflammation of the paranasal cavities, inflammation of the middle ear, catarrhal pneumonia of the aspiration-teletanic 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 per dose), with the antihistamine loratadine (3 mg per dose), ascorbic acid (300 mg per dose) and rutin (rutoside) (20 mg per dose) the anti-inflammatory activity of paracetamol 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 (vitamin C) is involved in redox processes, regulates these processes and carbohydrate metabolism, capillary permeability, blood coagulation, tissue regeneration and activates immune responses.

Upon receipt of HFS in the form of capsules or tablets, 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 because of the chemical structure of paracetamol (I- (4-hydroxyphenyl) acetamide, or paraacetaminophenol ) with a low content of technological standards of auxiliary substances, the mutual influence of the components, the conversion of paracetamol and other components of the composition into other chemical compounds is possible, and as a result, the biological eskoy (antiviral,

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In HFCs in the form of a powder, this danger is absent due to the introduction of a large amount (by weight) of auxiliary substances and the spread of the active ingredients over the entire mass of the powder.

Characteristics of biologically active drugs that make up the composition:

loratadine: pronounced antihistamine effect, prevents the development of tissue edema associated with the release of histamine;

calcium gluconate: prevents the development of increased permeability and fragility of blood vessels, causing hemorrhagic processes in influenza and acute respiratory viral infections, and also restores capillary circulation, has an antiallergic 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.

When all 6 ingredients are in one capsule / tablet and excipients that separate them little (about 3.5% of the total mass), chemical reactions between the constituent drugs during storage violate the overall composition of the dosage form, i.e. FPP is losing stability. Because of this, the composition was divided into 2 parts according to chemical compatibility, so the drug in the form of capsules / tablets is 2 capsules or 2 tablets.

As a result of studying the properties of a single composition of the claimed drugs and the selection of quantitative ratios of its active components, the optimal ratio of ingredients with a high therapeutic effect when they are taken together was found. Their weight ratio in OTC powder or with breakdown by OTC capsule / tablet, mg:

360 (included in capsule / tablet 1)

300

one hundred

Paracetamol

Remantadine

Ascorbic acid rutin

Loratadine

Calcium gluconate (the last 5 components are included in tablet 2).

In the manufacture of a dosage form (HFS) of this composition, each of the capsules / tablets included in the claimed composition contained pharmacologically and technologically acceptable excipients: capsule / tablet 1-20 mg, capsule / tablet 2-7 mg. In the SFS powder of excipients - 4167 mg.

The action of the proposed anti-influenza composition of the active components (in the experiments, PP is indicated) was tested in mice and rats. Acute and subacute toxicity, antiviral and interferonogenic effects of the proposed composition were evaluated. Acute toxicity of 1111 in mice was 1100 mg / kg with intraperitoneal administration. A study of subacute (within 15 days) toxicity of 1111 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, 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 showed that it has a pronounced protective therapeutic and preventive anti-influenza effect, which was revealed in a model of lethal influenza infection in white mice.

This effect is manifested in all studied indicators:

index of protection, the duration of the incubation period, macroscopic manifestations of a typical post-influenza chronic bronchopulmonary process typical of mice.

The mechanism of antiviral activity of the composition ίη νίνο 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, 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.

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

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 was 2-3 times. Duration of treatment - 3-5 days, improvement from

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The same results were obtained when patients were prescribed drinking (preferably hotter) based on the claimed composition in the form of an HFS powder for solution.

The high clinical efficacy of the complex drug is presented in table. one.

The proposed HFRS have an effect both as a means of symptomatic therapy and as a means affecting 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 effect of anti-influenza composition on the immune system in patients with acute respiratory viral infections

Index 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 1§A Mg / L 163 ± 13 172 ± 28 155 ± 23 174 + 21 * 1ёМ Mg / L 135 ± 8 134 ± 8 120 ± 12 154 + 17 * Mg / L 194 ± 8 200 ± 15 189 ± 14 20.5 + 7 *

Table 3

The effect of anti-influenza 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

* Differences with indicators before treatment are significant, p <0.01.

SFS - capsules / tablets

For industrial production, the composition in the form of OTC - capsules / tablets must be stable, meet the requirements of the pharmacopeia. The components are grouped by their chemical properties and by compatibility in 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 drug. 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.

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The quantitative composition of each of the capsules.

Capsule 1 (P): the average weight of the contents of the capsule is 380 mg, with 360 mg of paracetamol and 20 mg of excipients in it.

Starch (potato or gelatinized starch) 9.0

Milk sugar (lactose) 4.2

Magnesium stearate 3.8

Silicon Colloidal Dioxide 3.0

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

Thus, the composition of the capsule or tablet 1 represents the weight ratio of the components, mg: Paracetamol 324-396

Excipients are 18-22, and excipients consist of starch - 8.1-9.9 mg, milk sugar - 3.8-4.6 mg, magnesium stearate - 3.4-4.2 mg, colloidal silicon dioxide - 2.7-3.3 mg.

Capsule 2 (P): the average weight of the contents of one capsule is 480 mg, with the active ingredients in it - 473 mg and excipients - 7 mg.

The active components of the composition in this capsule, mg:

Remantadine (rimantadine) 50

Ascorbic Acid300

Rutin (Rutoside) 20

Loratadine3

Calcium Gluconate 100

As auxiliary substances used, mg:

Magnesium Stearate 4.8

Starch (potato) 2.2

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

Thus, the composition of the capsule or tablet 2 represents the weight ratio of the components, mg:

Remantadine 45-55 Vitamin C 270-330 Routine 18-22 Loratadine 2.7-3.3 Calcium gluconate 90-110 Excipients 6.3-7.7

moreover, excipients consist of magnesium stearate - 4.3-5.3 mg, starch (potato) 2.0-2.4 mg.

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 production of tablets is two different colors for each composition from the general composition.

The spread in the mass of the biologically active ingredients included in the composition while maintaining the full activity (drug effect) is ± 10%. Thus, in a single dose of 2 capsules / tablets, drugs are contained in the ratio, g:

Paracetamol Remantadine Ascorbic acid rutin Loratadine Calcium gluconate 0.324-0.396 0.045-0.055 0.270-0.330 0.018-0.022 0.0027-0.0033 0,090-0,110

The stability of the composition was checked after 12 months and after 24 months, the contents of each capsule (tablet) No. 1 and 2 were checked for compliance with the component (qualitative and quantitative) composition in accordance with the requirements of Pharmacopoeia GF XI, 1.2. Tested 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) - a mixture of powder and / or granules of yellow with a greenish tint and white.

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Table 4

The results of the stability check of the contents of the capsules No. 1 and 2 constituting the claimed composition

Party number The name of indicators Year of manufacture, 2002 Average weight in g Year of verification, 2003 Average weight in g Year of verification 2004 Average weight in g Color matching Number 1 Paracetamol 0.362 0.363 0.362 corresponds to one N ° 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 Number 1 Paracetamol 0,357 0.356 0,358 corresponds to 2 Number 2 Vitamin C 0.297 0.299 0.298 Calcium gluconate 0,101 0,100 0.102 Remantadine 0.051 0,052 0.051 corresponds to Routine 0,021 0,020 0,021 Loratadine 0.0031 0.0030 0.0031 Number 1 P Ί / ΙΟ η all η alA. ιΐίψαμνΛ siukam s Number 2 Vitamin C 0.294 0.296 0.296 Calcium gluconate 0.104 0.104 0.102 Remantadine 0,053 0,052 0.051 corresponds to Routine 0.019 0.019 0,020 Loratadine 0.0028 0.0029 0.0028 Number 1 Paracetamol 0.348 0.349 0.350 corresponds to 4 Number 2 Vitamin C 0.310 0,309 0,307 Calcium gluconate 0,092 0,094 0,096 Remantadine 0,049 0,049 0,050 corresponds to Routine 0.019 0,020 0.019 Loratadine 0.0031 0.0031 0.0030 Number 1 Paracetamol 0.364 0.363 0.364 corresponds to Number 2 Vitamin C 0.295 0.297 0.297 Calcium gluconate 0.103 0.102 0.102 Remantadine 0,052 0,050 0,049 corresponds to 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 HFRS - capsules / tablets is possible only in the form of different capsules or tablets having a chemically compatible composition.

GLS-powder for solution for administration Reg о§

SFS powder is prepared by mixing dry active and auxiliary components or granulating them with subsequent packaging in a bag (contour bezel-free packaging).

During the manufacturing process, deviations in the mass of the contents of the packets are permissible and amount to ± 10%. Active substances, mg:

Paracetamol 360 Calcium gluconate one hundred Remantadine fifty Vitamin C 300 Routine twenty Loratadine 3

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Excipients, mg:

Aspartame30

Oxypropyl methylcellulose 10

Fragrance60

Silicon Colloidal Dioxide (Aerosil) 20

Lactose (milk sugar) 4047

Lactose is an excipient in pharmaceutical preparations; this disaccharide is a substance stabilizing the composition.

Aspartame is a methylated dipeptide of two amino acids: aspartic and phenylalanine, used as a sweetener.

Aspartame is part of the claimed composition - powder, since it is acceptable for patients with diabetes mellitus, and there are no restrictions on the appointment of an anti-influenza drug.

Oxypropyl methylcellulose (HMPC) is included as a binder in the OTC powder.

In industrial production, the composition of the powder is usually introduced to remove the electrostatic charge of aerosil. As a "sliding" auxiliary substance that prevents the mixture from electrifying, Aerosil is included in the inventive GLS powder.

To give the solution a pleasant taste and commercial attractiveness of the drug, dry flavoring is included in the SFS powder. Flavors that are added to the powder have a taste of honey, or lemon, or blackcurrant, or other berries or fruits. Such flavors are approved for use in the pharmaceutical industry.

SFS powder was tested for stability in accordance with the requirements of pharmacopoeia GF XI, 1.2. The composition was qualitatively and quantitatively checked after 12 and after 24 months. All values of the tested components of the mixture were normal. The results of the verification of 2 batches are given in table. 5.

Table 5

Anti-Influenza Powder Stability Test Results

Party number The name of indicators Year of manufacture 2002 average weight in mg. Year of verification, 2003 average weight in mg Year of verification, 2004 average weight in mg Color matching one Paracetamol Ascorbic Acid Calcium Gluconate Remantadine Rutin Loratadine 356 299 one hundred fifty 21 health 355 297 one hundred fifty twenty 3.0 356 298 101 51 21 3,1 Compliant 2 Paracetamol Ascorbic Acid Calcium Gluconate Remantadin Routine Loratadine 371 305 101 52 twenty 2.9 370 303 102 51 21 2.9 370 304 one hundred 51 twenty 2.9 Compliant

As a result of tests on the stability of the HFS powder, it was found that changes in the composition do not occur during storage even after 24 months.

The excipients introduced into the composition make the composition highly soluble in water; during storage, its organoleptic properties do not change.

The permissible spread in the mass of excipients is ± 10%. Therefore, a single dose of an HFS powder contains the active substances, mg:

Paracetamol 324-396 Remantadine 45-55 Vitamin C 270-330 Routine 18-22 Loratadine 2.7-3.3 Calcium gluconate 90-110 excipients, mg: Lactose 3642-4452 Aspartame 27-33 OPMC 9-11 Aerosil 18-22 Flavoring 54-66

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The total mass of the contents of the SFS powder is 5000 mg ± 10%, i.e. 4500-5500 mg.

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 (1111) was a yellowish powder, relatively poorly soluble in water. The known anti-influenza drug remantadine and the thiobenzimidazole-almide derivative were used as comparison preparations.

Animals.

Experimental studies were performed on white mongrel mice weighing 18-22 g, obtained from the Rappolovo nursery of the Russian Academy of Medical Sciences. 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 AyUY / 2/68 (Η3Ν2), 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 introduction of drugs was carried out in test doses, which are shown in table. 5, orally (through a probe) in a volume of 0.2 ml five times according to the following scheme: 24 and 1 hours before infection, 24, 48 and 72 hours after infection. The control group mice were similarly injected with a 1% starch solution. Mice were infected intranasally with the pathogenic influenza virus Α / Αίο1ύ / 2/68 under mild ether anesthesia at a dose of 0.1 and 1 L0 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 according to the presence of 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% - lesion of individual lobes , 100% total lung lesion.

Statistical processing of the results of macroscopic studies was carried out by comparing confidence intervals of arithmetic mean values 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. 6. 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:

143 _ KZ - 1 x 100 - percentage of dead mice in control KZ; percentage of dead mice in the experiment where KZ is the coefficient or ratio of protection.

The results show that the calculated value of IZ for 1111 and almide at an infectious dose of 1 L0 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 injected with 1111 and almide, at an infectious dose of 1 L0 ₅₀ , the incubation period increased by 1.8-2.2 days compared to the control. However, its greatest increase (by 3.5 days) was observed with the introduction of remantadine.

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Table 6

The influence of test drugs on the course of experimental influenza infection in mice

Samples of the drug, dose, mg / kg Number of animals in R ruble The dose of the virus, PO50 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 -n- nineteen 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 + -G + PP, 100 mg / kg nineteen 0.1 2 / 10.5 0,0 - twenty one 5 / 25.0 54.5 ++ Remantadine 50 mg / kg twenty 0.1 0 1000 + -H- + twenty one 1 / 5.0 90.9 1 1 and Control, placebo twenty 0.1 2 / 10.0 - - twenty one 11 / 55.0 - -

* 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 14E _{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 7

Calculation of the incubation period for the entire group of animals

Drug, dose, mg / kg Yu virus dose 50 Number of mice Of these, fell on the indicated days of the experiment. Total fell The incubation period, days 4 5 6 7 8 nine 10 eleven 12 thirteen 14 Almide, 10 mg / kg 0.1 twenty - one - - - one - - - - 2 13,4 one nineteen 2 one - 2 one - one - - 6 12.3 PP, 10 mg / kg 0.1 twenty - - - - one - one 13.7 one twenty - one 2 - one - 4 12.6 PP, 100 mg / kg 0.1 nineteen - - - one one one 3 13.7 one twenty - 2 2 - one - 5 12,2 Remantad in, 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 lesions of the lungs on the 14th day of influenza infection is 30% with the introduction of diamond.

PP (10 mg / kg) - 20%,

PP (100 mg / kg) - 35%, herbal medicine (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 mechanism of antiviral activity of these compounds ίη νίνο may be due to the induction of serum interferon, which was found in mice after administration of these drugs. This is consistent with literature data 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 to com

-9008765 Complex prevention and treatment of influenza and other viral infections.

The study of the interferogenic action of AI

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 Vaccines 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. The anti-influenza drug is a yellowish powder, poorly soluble in water. As a comparison drug used a derivative of thiobenzimidazole almide. Doses of the preparations were 10 and 100 mg / kg for NI and 10 mg / kg for almide. Test samples were previously 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 performed on white mongrel mice weighing 18-22 g, obtained from the Rappolovo nursery of the Russian Academy of Medical Sciences. Mice were divided into 5 groups of 25 animals each. In total, 110 animals were used in this work.

Interferon Testing Method.

Interferon was tested according to the method Гш1ег Ν.Β. (1968) modified by 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 forms: 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 min, washing was performed from cells not adsorbed onto the formation with a 4-fold phosphate-buffered solution, and dried for 10 min in an incubator at a temperature of 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 orthophenylene-diamine. 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., on the activity of the Sendai virus. 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, which reflects the onset of a decrease in the testvirus activity (Table 8).

Table 8

Calculating the working dose of Sendai test virus

Testvirus Activity in the enzyme immunoassay of the 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 No 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 1: 200 virus dilution. This dilution of the virus was introduced into the main experiment with the test sera for a day at 37 ° C. After 24 hours, all the test material was washed in the same way as when 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 min and a 2-day treatment with 1Ν hydrochloric acid.

The results of the study.

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, II - 10 and 100 mg / kg. The cycle of drug administration was 3 days. 1, 2, 3, 4, 5 days after the first injection of the drug was taken

- 10 008765 blood from 5 mice of each group and received serum. 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 dose of PP, 10 mg / kg stimulated the production of interferon to a concentration of 76.3 units / ml, after a dose of HH 100 mg / kg, up to 120.6 units / ml (Table 9).

When evaluating the production of alpha interferon (Table 10), it was found that almide 10 mg / kg stimulated the production of alpha interferon for 3 days after oral administration to 40.5 units / ml, in the future its activity dropped to 17, 9 units / 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 9

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 oral administration one 2 3 4 5 Almide, 1 0 mg / kg 28,2 44.3 4.05 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 10

The concentration of alpha-interferon in the blood of mice after oral administration of test drugs

Drugs, dose, mg / kg Interferon titer in the blood of mice, days after the first injection one 2 3 4 5 Almide, 1 0 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 11), 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 5th day it was 30.5 units / ml with a dose of 10 mg / kg and with another dose of 100 mg / kg - 40, 0 units / ml.

Table 11

The concentration of gamma 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 4 5 Almide, 1 0 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 3 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 5th 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 (5)

1. An anti-influenza complex drug for oral administration, consisting of paracetamol, remantadine, ascorbic acid, rutin, loratadine, calcium gluconate, in the following ratio of components per dose by weight, mg: Paracetamol Remantadine Ascorbic acid Rutin Loratadine Calcium gluconate 2. The anti-influenza complex drug according to claim 1, characterized in that it further comprises auxiliary substances for the preparation of the finished drug in the form of capsules or tablets, and their mass is 24.3-29.7 mg in a single dose. 3. The anti-influenza complex drug according to claim 1, characterized in that it further comprises auxiliary substances for the preparation of the finished drug in the form of a powder for the preparation of a solution for drinking, and their mass is 3750-4584 mg in a single dose. 4. The anti-influenza complex drug according to claim 2, characterized in that the active components are separated by chemical compatibility into two capsules or tablets, which together comprise a single dose, and capsule 1 or tablet 1 contains paracetamol and excipients, mg: Starch 8.1-9.9 Milk Sugar 3.8-4.6 Magnesium stearate 3.4-4.2 Colloidal silicon dioxide 2.7-3.3 capsule 2 or tablet 2 contains remantadine, ascorbic acid, rutin, loratadine, calcium gluconate and excipients, mg: Magnesium Stearate 4.3-5.3 Starch 2.0-2.4 5. Anti-influenza complex drug according to claim 3, characterized in that it is made in the form of a powder for the preparation of a solution for drinking, containing as auxiliary substances, mg: Aspartame Hydroxypropyl methylcellulose Flavoring Colloidal silicon dioxide Lactose 27-33 9-11 54-66 18-22 3642-4452 Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2/6