RU2687745C1 - Combined drug preparation in form of solution for preparing spray for treating diseases of oral cavity - Google Patents

Abstract

FIELD: medicine; pharmacology.SUBSTANCE: invention refers to pharmacology and concerns a drug for treating oral diseases. What is presented is a medicinal agent in the form of a solution for preparing a spray for treating oral diseases, which contains a combination of two active components, excipients and a solvent. First active component is branched oligohexamethylene guanidine of formula,where R isor,n, nand nare equal to 1–3, a z is equal to 0.15–1.10 with molecular weight distribution M/Mfrom 5.4 to 9.3 with weight-average molecular weight Min the range from approximately 3,800 to 6,300 and number-average molecular weight Min the range from approximately 600 to 1100 in form of hydrochloride (OGMG-GC). Second active component is selected from benzalkonium chloride or benzoxonium chloride. Excipients include glycerol, menthol, sodium saccharinate, and the solvent is purified water or a mixture thereof with ethyl alcohol. Medicinal agent has the following composition (wt/vol. %): Osmag-GC 0.1–0.5; benzalkonium chloride 0.005–0.05 or benzoxonium chloride 0.01–0.02; glycerine 30–50; menthol 0.01–0.03; sodium saccharinate 0.03–0.06; solvent - up to 100.EFFECT: proposed drug is effective against a wide range of pathogenic microorganisms and can be used in dental and ENT practice.1 cl, 8 tbl, 10 ex

Description

The technical field to which the invention relates.

The invention relates to the field of pharmacology. More specifically, it provides the drug in the form of a solution to obtain a spray for treating diseases of the oral cavity, such as lesions of its mucosa (glossitis, gingivitis, stomatitis, alveolitis, periodontitis), containing a combination of two active ingredients, excipients and a solvent. The invention can find application in the dental and ENT practice.

The level of technology

One of the most common causes of inflammatory processes is the influence of pathogenic microflora. In the field of dentistry, diseases of the oral mucosa (glossitis, gingivitis, stomatitis, periodontitis) are diseases caused by it. Official statistics indicate an increasing incidence of such diseases: gingivitis (inflammation of the gums) and periodontitis (inflammation of the periodontal ligament structure and bones of the alveolar processes) make up 94-96% of all periodontal diseases, and already in childhood the prevalence of gingivitis reaches 80-95%.

One of the problems that practitioners face is the acquisition of resistance to existing drugs by pathogenic microorganisms. For example, some types of staphylococci have become resistant to aminoglycosides, in particular - to gentamicin. This makes actively used drugs ineffective, or causes the need to increase their doses. The latter circumstance is associated with an increase in toxicity and the likelihood of side effects. Also, the treatment of diseases of this kind is also complicated by the fact that both aerobic and anaerobic forms of bacteria are the causative agents of the infectious process. All this necessitates the creation of new pharmaceutical compositions with a broad spectrum of antimicrobial action, to which the pathogenic microflora has not developed resistance.

A possible approach is the creation and use of compounds acting by a different mechanism than antibiotics. In particular, a known group of copolymer cationic polyelectrolytes is derivatives of guanidine, biguanidine or biguanide, which cause disruption of the structure and integrity of bacterial membranes, which leads to disruption of the correct ion balance between the cytoplasm of the cell and the external environment, the output of the cell contents to the medium, loss of the ability to maintain osmotic pressure and, ultimately, to lysis. It is also possible the penetration of environmental components into the cell.

In particular, for polyhexamethylene guanidine hydrochloride (PHMG-GC) by electron microscopy it was shown that it damages the cell membrane of E. coli (Zhou ZX et al. Damage of Escherichia coli by microhexamethylene guanidine hydrochloride: Micrographic evidences // J. Appl. Microbiol. 2010. Vol. 108. No. 3. Pp. 898-907). Cells treated with 23 μg / ml PHMG collapse and lose their characteristic cylindrical shape, and the substrate is covered with a large amount of debris. The images obtained using transmission electron microscopy also show the destruction of the outer membrane of E. coli and the condensation of cell contents. In addition, in the same work it was shown that PHMG-GC promotes cell loss not only of DNA and RNA, but also of proteins: under the action of PHMG-GC, β-galactosidase, normally contained in the cytoplasm of E. coli, is released into the solution.

Similar changes in the structure of E. coli cells after exposure to PHMG-GC were shown using atomic force microscopy in Qian L. et al. AFM and CLSMI / ACS Appl. Synthesis of modified guanidine-based polymers and their antimicrobial activities. Mater. Interfaces. 2011. Vol. 3. No. 6. Pp. 1895-1901. In addition to morphological changes, the authors also showed that PHMG-GC promotes the penetration of fluorescent dye, propidium iodide, inside cells, which is normally not able to pass through intact cell membranes.

Despite the fact that the mechanism of action of compounds - derivatives of guanidine is described in some detail in the literature, all the above-mentioned studies were carried out on E. coli bacteria. However, the mechanism of action of such compounds against gram-positive microorganisms, as well as anaerobic organisms, has not been established. In addition, the medical use of PHMG-GC is hampered by the presence of residual highly toxic hexamethylene diamine (HMDA). Therefore, the main areas of application of PHMG-GC are cleaning and / or disinfection of residential and public facilities, disinfection of medical equipment, surfaces and textiles in hospitals, woodworking, agriculture, veterinary medicine, water treatment and water treatment, the fight against pathogenic microorganisms in ventilation and air conditioning systems , during storage and transfer of petroleum products.

Attempts were made to remove HMDA both after the completion of the synthesis of PGMG-GC and as its stage. The latter approach is implemented, in particular, in the invention in accordance with the application CN 105820333 (publ. 03.08.2016). The method disclosed in it involves the polycondensation reaction, after which the saponification reaction is carried out: at a temperature of the reaction mass of 140-160 ° C, add sodium hydroxide solution to pH 12 and above, after the addition of sodium hydroxide is maintained, the temperature is maintained within 90-120 ° C for 1 hours or longer to produce water-insoluble polyhexamethylene guanidine, which is then transferred to the hydrochloride form by dissolving in hydrochloric acid and dried. However, the purity of the product obtained is at the level of 96%, which is lower than acceptable for a pharmacopoeial substance.

Information on medical applications of PHMG-GC is very limited. Thus, patent RU 2143905 (publ. 10.01.2000) discloses the use of polyhexamethylene guanidine in the form of hydrochloride, phosphate, benzoate, gluconate as drugs with antimicrobial activity against anaerobic and mixed infections, represented by Staphylococcus spp., Proteus spp., Pseudomonacee, ., Clostridium spp., Porphiromonas spp., Prevotella spp. and others, i.e. possesses a wide range of antibacterial action. The description states that "The invention relates to medicine and can be used in surgery, dentistry, gynecology, urology, therapy, and other areas where anaerobic or mixed infection is possible". In the examples, the activity is determined in vitro for salts pgmg linear structure, which follows from depicted in the description of the structural formula:

where, n = 5-15, y = hydrochloride, phosphate, gluconate, benzoate. Thus, the invention only confirms a wide range of antibacterial activity of PHMG, but does not contain specific indications of possible formulations for use in dentistry and ENT-practice, as well as on combined preparations.

Known branched copolymers of guanidine and hexamethylenediamine, hereinafter referred to as oligohexamethylene guanidines (OGMG). In the patent RU 2443684 (publ. 27.02.2012) disclosed branched oligomers hexamethylenediamine and guanidine of the formula (I)

where R is

and n ₁ , n ₂ and n ₃ are 1-3, az is 0.15-1.10 with a molecular mass distribution of M _w / M _n from 5.4 to 9.3, with a mass-average molecular weight M _w in the range from about 3800 to 6300 and a number average molecular weight of M _n in the range of from about 600 to 1100, as a salt. The method of their synthesis, disclosed in the exemplary embodiment of the invention, does not include a purification step, since, in accordance with the invention, they are intended to produce a disinfectant. However, the description states that the preparation of the base can be carried out in the presence of an alcohol, such as ethanol or isopropanol, taken in an amount of 0.5-1.0 volume of the total volume of the reaction mass, consisting of equal volumes of approximately 50% aqueous solutions of oligomer and alkali. In this case, the residual content of HMDA can be reduced to 0.02-0.1% of the mass. depending on the time and temperature of the process, as well as the ratio of reagents and alcohol. A product of this purity is quite suitable as a pharmaceutical substance.

The patent RU 2513997 (publ. 04/27/2014) discloses an ophthalmologic drug in the form of droplets containing a combination of active ingredients, a promoter component, excipients and water, characterized in that the combination of active ingredients consists of the branched polyhexamethyleneguanidine of formula (I) shown above, where R, n ₁ , n ₂ and n ₃ , z, M _w / M _n , M _w and M _n have the above values, present in the form of hydrochloride, and taurine; the promoting component is selected from succinic acid or its pharmaceutically acceptable salt; the excipients are selected from the group consisting of physiologically tolerated alkaline or acidic agents present in amounts required to maintain the pH of the preparation in the range from 6.0 to 7.8, and salt tonic an agent selected from physiologically tolerable salts of sodium or potassium and their mixtures, present in an amount that provides the tonicity of the drug, comparable to the tonicity of the natural tear fluid is healthy wow man. The drug has a wound healing effect in case of eye injuries. In vitro, its bactericidal action against E. coli, Staphylocuccus spp. and Candida spp., however, the description of the invention does not contain data on the effective treatment of bacterial ophthalmic infections. The subject invention shows the bactericidal effect of OGMG-GC on E. coli, Staphylocuccus spp. and Candida spp., which may be present in the oral cavity, but refers to ophthalmic and not dental drugs.

A similar note applies to patents RU 2509562 and RU 2510264. Patent RU 2509562 (published March 20, 2014) is a drop-on ophthalmic drug containing polyhexamethyleneguanidine, phosphate-buffered saline, a hydrating component selected from the group consisting of dextran, a copolymer of vinylpyrroline, a copolymer of vinylpyrroline; / vinyl acetate and water-soluble methylcellulose, characterized in that polyhexamethylene guanidine is represented by branched oligomers of formula (I) shown above, where R, n ₁ , n ₂ and n ₃ , z, M _w / M _n , M _w and M _n have the above values are present Enikeev the hydrochloride, and the formulation further comprises a copolymer based on N-vinylpyrrolidone general formula (II):

where the monomer unit M is a fragment of 2-methyl-5-vinyltetrazole (MW) or 2-methyl-5-vinylpyridine (MVP):

and the content of monomer units n is 25-50 mole. %, viscosity-average molecular weight M _μ copolymer depends on the nature of M: if M is MW, then M _μ = 30-50 kDa; if M represents the profit center, then M _μ = 15-40 kDa. The use of the invention allows to increase the effectiveness of treatment of dry eye syndrome, as well as possible concomitant bacterial and fungal infections caused, in particular, C. albicans. The drug does not irritate with prolonged use.

In the patent RU 2510264 (publ. 03/27/2014) the ophthalmologic preparation in the form of drops is also presented. But, in contrast to the patent RU 2513997, in the combination of the active components instead of taurine is sulfacetamide. The drug on the dog model showed activity against E. coli, C. albicans, P. aeruginosa, S. oralis, which allowed to cure purulent conjunctivitis.

Various mechanisms of action of benzalkonium chloride and benzoxonium chloride have been proposed; however, the most likely are the disturbance of redox processes in the microbial cell by reacting with the amino groups of the proteins of microorganisms. They also suggest that they inhibit certain bacterial enzymes. In particular, it has been established that benzalkonium chloride suppresses plasmocoagulase and staphylococcal hyaluronidase. Thus, the indirect membranotropic effect is realized, i.e. action of the active substance indirectly through interference with the cytoplasmic metabolism or by other indirect means.

In patent RU 2219906 (publ. 12/27/2003) an invention discloses an aqueous drug preparation in the form of a solution containing a pharmacologically active substance to obtain aeropentless aerosols for inhalation, characterized in that it contains a complexing agent in a concentration of from 10 to 100 mg / 100 ml The active substance is selected from the group of beta mimetics, anticholinergics, antiallergic drugs and / or antihistamines. The agent in question has been designed for other uses determined by the active substance, and benzalkonium chloride is used as a preservative, and not as an active ingredient.

In the application KR 20170033925 (publ. 03/28/2017) disclosed, in particular, antiviral and antibacterial composition in the form of a spray, obtained by mixing the main component with 1-10% benzalkonium chloride and adding 1-3% hydrocortisone, mixing and diluting 30-150 mass parts of the mixture per 100 mass parts of the main component; 100-100000 mass parts of purified water per 100 mass parts of the mixture. Composition useful for the treatment of respiratory diseases caused by viral and bacterial infections, has increased efficiency, but is aimed at solving a different task than the present invention.

In published international application WO 00/047203 (publ. 08/17/2000), a composition effective for delivering into the oral cavity an effective amount of at least one pharmaceutical agent in admixture with an oral absorption enhancer in an orally acceptable carrier solvent, where the oral enhancer is disclosed absorption is adapted to modify the surface membrane of the patient’s oral cavity in order to improve the oral absorption of the pharmaceutical agent. Examples of the pharmaceutical agent, in particular, are glucocorticoid steroids, dexamethasone, prednisone, barbiturates, benzodiazepines, sedatives, peptides and proteins up to 50 kDa, non-narcotic and narcotic analgesics. The oral absorption enhancer can be selected from hydroxypropyl-β-cyclodextrin, benzalkonium chloride, benzethonium chloride, polysorbate 80, sodium lauryl sulfate, Tween, Brij, Pluronic. The invention provides a quick, painless and toxicologically safe administration of the active substance. The purpose of benzethonium chloride differs from that in the present invention.

In published international application WO 2002/002128 (publ. 25.04.2004) discloses a topical oral composition in the form of a spray, comprising a safe and effective amount of antimicrobial agent such as triclosan, chlorhexidine, benzalkonium chloride, salicylanilide, cetylpyridinium chloride, tetradecyl pyridinium chloride and pharmaceutically acceptable carrier. The composition is active against P. gingivalis, B. forsythus, A. actinomycetemcomitans, T. denticola, T. socranskii, F. nucleatum, P. intermedia, L. acidophilus, L. casei, A. viscosus, S. sobrinus, S. sanguis, S. viridans and S. mutans, which allows its use for the treatment of gingivitis and periodontitis. The composition contains the only active ingredient.

International application WO 2013/063695 (published 10.05.2013) discloses a composition for the prevention and treatment of diseases of the oral cavity, including (a) an iron-binding protein selected from ovotransferrin, lactotransferrin and serotransferrin, (b) a chelating agent selected from EDTA, EGTA, DTPA, EDDHA, IDA, CDTA, HEDTA, HEIDA, NTA, sodium citrate, potassium citrate and zinc citrate. It also contains an infection control compound selected from a broad group, including, in particular, benzalkonium chloride. The composition is active against Veillonella spp., Actinomyces spp., Bacillus spp., Mycobacterium spp., Fusobacterium spp., Streptococcus spp., Enterococcus spp., Prevotella spp., Porphyromonas spp., Clostridium spp., Acineto chtophteph, sine. , K. pneumoniae, E. coli, P. cangingivalis and C.albicans and can be presented as a spray.

There is evidence that pathogens such as Pseudomonas aeruginosa develop resistance to benzalkonium chloride by increasing the content of phospholipids, as well as fatty and neutral lipids in cell walls (Sakagami Y., Yokoyama N., Nishimura H., Ose Y ., and Tashima T. Mechanism of resistance to benzalkonium chloride by Pseudomonas aeruginosa // Appl. Environ. Microbiol. 1989 Aug. Vol. 55 (8). Pp. 2036-2040.

From the patent RU 2437647 (published on 12/27/2011) a pharmaceutical composition with antibacterial, antiviral and antifungal effects is known, containing chlorhexidine and benzoxonium chloride, water, wherein chlorhexidine and benzoxonium chloride are used as the main active substances, the purified water is used as solvent and shaper in the following ratio of components, wt./about. %:

Chlorhexidine 0.1-1.0 Benzoksoniya chloride 0.05-0.5 Purified water the rest is up to 100.

The composition is indicated to be synergistic. It is used in the form of a no-spray spray for antiseptic treatment and disinfection of wounds, burn surfaces, skin cracks, damaged mucous membranes and insect bites, but the data confirming the implementation of at least one of these appointments does not contain a description of the invention.

There is a TeraFlu® LAR spray for local use on the pharmaceutical market (Manufacturer: Novartis Consumer Health SA., Switzerland), which is a clear, colorless mint smell solution that contains (by weight / volume%) active substances - benzoxonia chloride - 0.2% and lidocaine hydrochloride - 0.15%, and excipients:

ethanol 96% (v / v) - 10%; glycerin - 15%; hydrochloric acid 0.1 n - 0.0581%; peppermint oil - 0.01%; menthol - 0.0025%; purified water - up to 100%. The official instructions for use (available at the link: https://medi.ru/instrukciya/teraflyu-lar-sprej_11160/) indicated that benzoxonium chloride is a quaternary ammonium salt [N-benzyl-N-dodecyl-N, N-di (2-hydroxyethyl) ammonium chloride], due to its cationic structure, it has membranotropic activity and has a pronounced antibacterial effect against gram-positive and, to a lesser extent, gram-negative microorganisms. Lidocaine is a local anesthetic that during inflammation reduces pain in the throat when swallowing. The indications are infections of the oral cavity and pharynx: pharyngitis, laryngitis, catarrhal sore throat, stomatitis, ulcerative gingivitis.

Thus, in the prior art, no solutions have been identified that provide for the treatment of infections of the oral cavity that are similar in composition and principle of action with the proposed combination drug.

Disclosure of the invention

An object of the invention is the creation of a drug in the form of a solution to obtain a spray for the treatment of diseases of the oral cavity, caused, in particular, by a bacterial infection that does not cause resistance and is effective against a wide range of pathogenic microorganisms in order to expand the Arsenal of means of the specified destination for substitution in the market imported products.

As a result of research, the inventors have found that the technical problem can be solved by creating a drug in the form of a solution to produce a spray for treating diseases of the oral cavity, containing a combination of two active ingredients, excipients and a solvent, in which the first active ingredient is a branched oligohexamethyleneguanidine of the formula

where R is

n ₁ , n ₂ and n ₃ are 1-3, az is 0.15-1.10 with a molecular mass distribution of M _w / M _n from 5.4 to 9.3 with a mass-average molecular weight M _w in the range from approximately 3800 to 6300 and a number average molecular weight of M _n in the range from about 600 to 1100 in the form of hydrochloride (OGMG-GC), and the second active ingredient can be selected from benzalkonium chloride

or benzoxonia chloride

excipients include glycerin, menthol, sodium saccharinate, and the solvent is purified water or its mixture with ethyl alcohol.

Preferably, the drug has a composition (w / v%):

OGMG-GH 0.1-0.5; benzalkonium chloride 0.005-0.05; or benzoxonia chloride 0.01-0.02; glycerol 30-50; menthol 0.01-0.03; sodium saccharinate 0.03-0.06; solvent up to 100.

More preferably, the drug has a composition (w / v%):

OGMG-GH 0.1-0.3; benzalkonium chloride 0.005-0.05; glycerol 30-40; menthol 0.01-0.025; sodium saccharinate 0.03-0.05; solvent up to 100.

In contrast to antibiotics, the proposed drug does not cause the emergence of resistant strains of microorganisms. It also has low toxicity and allergenicity, is not mutagenic, carcinogenic and teratogenic. Acute intragastric toxicity for outbred rats is 450 mg / kg (female) and 500 mg / kg (male). It is not possible to achieve a dose higher than 370 mg / kg with irrigation of the rats oral cavity, however, with such a dose, there are no signs of acute toxicity. Signs of chronic toxicity are absent at a concentration of 15.6 mg / kg, which corresponds to a 20-fold therapeutic dose (TD). The highest daily therapeutic dose (TD × 10) recommended for humans is 0.13 mg / kg. The highest daily therapeutic dose for rats with an average body weight of 280 g is 0.77 mg / kg, and for mice with a body weight of 25 g, it is 1.5 mg / kg.

In the Ames mutation test on Salmonella typhimurium (TA 100, TA 98 and TA 97 strains), in the test for micronuclei in polychromatophilic erythrocytes in the bone marrow of mice and in the test for chromosomal aberrations in human lymphocytes of peripheral blood, studies showed that the test drug was not mutagenic ( under the full program) and cancerogenic (in short-term testing) activities.

For intraperitoneal administration of the drug in a test of general anaphylaxis at a dose of 6.0 mg / kg, the Weigle index is 1.6 for males and 1.8 for female guinea pigs, which is characterized by mild anaphylactic shock (moderate anaphylactic shock is characterized by a Weigle index from 2 to 3).

The teratogenicity of the drug during the irrigation of the oral cavity of both males and female outbred rats (7.5 mg / kg, TD × 10) was evaluated according to the main factors of the development of the offspring: body weight from 4 to 40 days, opening time of the eyes, eruption of incisors, otlichanie auricle, the appearance of coat, lowering of the testes (opening of the vagina), as well as the rate of maturation of the sensory-motor reflexes during the feeding period. All indicators in the groups of males and females were not statistically significantly different from control animals.

The drug is effective against a wide range of aerobic and anaerobic pathogens, such as, for example, Streptococcus spp. (S. oralis, S. pneumoniae, S. pyogenes), Staphylococcus spp. (S. aureus), Klebsiella spp. (K. pneumoniae), Pseudomomas aeruginosa, Escherichia coli, Peptostreptococcus anaerobius, Veillinella parvula, Porphyromonas gingivalis. In the course of the research it was found that the minimum inhibitory concentrations (BMD) are at levels: for Moraxella catarrhalis - 0.05 μg / ml, for S. pneumoniae and S. pyogenes - 0.25 μg / ml, Neisseria gonorrhoeae and S. aureus - 1 µg / ml.

For a medicinal product containing OGMG-GH at a concentration of 0.1% w / v, on the model of thermal traumatic stomatitis and gingivitis in rabbits, 7 days after the creation of the defect, there is an almost complete healing of the oral mucosa without signs of erosive-ulcerative lesions, inflammatory infiltration and hemorrhages.

The drug has a shelf life of 3 years, which is less than that of TeraFlu® LAR (5 years) on the market. However, this fact should not be considered a significant drawback of the proposed medicinal product, taking into account its undoubted advantages: a higher activity and the breadth of the spectrum of antibacterial action. In particular, on a model of traumatic stomatitis and gingivitis in rabbits, the authors found that a drug prepared in accordance with one embodiment of the invention contributes to wound healing at a dose of more than one and a half times less than TeraFlu® LAR.

Also in accordance with the invention, a mechanical device for spray-free production of a spray is proposed, comprising a drug reservoir and a spray unit sealed to it, connected along the drug flow to the reservoir with an intake tube, and connected to the atmosphere through the air flow. Preferably, the tank is made of an organic polymer of medical use, metal or glass, and the spray unit is driven by a limited course of its drive in the direction of the tank. This ensures the receipt of portions of the spray of a given volume, which protects against overdose. In accordance with this, aluminum monoblock 40 ml cylinders with an internal lacquered coating with a microdosing unit and a protective cap made of high-density polyethylene are preferred.

Further, the possibility of carrying out the invention with the achievement of its technical results will be shown in non-limiting examples.

The implementation of the invention

Examples 1-4. The preparation of the medicinal composition to obtain a spray

General method. The first active ingredient - OGMG-GC (m1 g, purity 99.8%, humidity 1.5%) is added to 50 ml of purified water with stirring, and after its dissolution, the second active component is added to benzalkonium chloride (BAC, m2 g) or benzoxonia chloride (BOC, m2 * g). To the resulting solution of the active substances add excipients: sodium saccharinate (m3 g), menthol (m4 g) and glycerin (m5 g). The resulting mixture is stirred for 15-20 minutes, after which its volume is adjusted to 100 ml by adding purified water. Then, the obtained medicinal composition is again thoroughly mixed for 5-10 minutes and is packaged in aluminum monoblock cylinders (40 ml) with an internal lacquered coating with a microdosing unit and a protective cap of high-density polyethylene.

The compositions of the prepared sprays (mass values of the components m1 ... m5 numerically coincide with their contents in wt./vol.%) Are given in table 1.

Example 5. Evaluation of acute toxicity of the drug

A) Assessment of acute toxicity after intragastric administration

Acute toxicity is estimated by the results of research on outbred rats (females and males) with an initial body weight of 210-260 g females, 300-350 g males. Animals are divided into groups (n = 5) by random sampling. The body weight is taken as a criterion. The variation in the initial mass does not exceed 20%. In addition, they form similar groups of control animals of each species and sex, with which a solvent is introduced in a similar way.

Animals are kept in polycarbonate cages with steel lattice lids with feed attachments (compound feed for laboratory rodents PK-120) and water. Animals receive food and water without restriction. Temperature 18-22 ° C, relative humidity 50-65%, twelve-hour lighting cycle and tenfold change of room air volume per hour. The drug prepared in accordance with example 3, is administered on an empty stomach intragastrically once through a metal atraumatic probe. Euthanasia decapitation with pre-anesthesia produced on the 14th day of the experiment. An autopsy is performed on the same day.

Evaluation of the toxic effect of the drug is carried out according to the following clinical features: 1) respiratory parameters (difficulty breathing, cyanosis, rapid breathing, nasal discharge); 2) motor activity (increased / decreased, drowsiness, loss of balance, sensitivity, catalepsy, ataxia, unusual movements, prostration, tremor, fasciculation; 3) convulsions (clonic, tonic, tonic-clonic, asphyctic); 4) reflexes (corneal, balance, myotatic, light, startle reflex); 5) eye signs (lacrimation, miosis, mydriasis, exophthalmos, ptosis, clouding, iritis, conjunctivitis, chromodotriorea, weakening of the flashing membrane); 6) cardiovascular events (bradycardia, tachycardia, arrhythmia, vasodilation, vasoconstriction); 7) increased salivation; 8) condition of coat (piloleiomyoma, alopecia); 8) analgesia; 9) muscle tone (hypotension, hypertension); 10) gastrointestinal indices (soft stools, diarrhea, vomiting, polyuria, rhinorrhea).

Skin, lymph nodes, aorta, heart, larynx, trachea, lungs, thymus, esophagus, stomach, small intestine, large intestine, liver, pancreas, spleen, kidneys, adrenal glands, bladder, testicles, ovaries, uterus, submandibular salivary gland lymph nodes, the thyroid gland, the brain is subjected to macroscopic examination. The heart, lungs, thymus, liver, spleen, kidneys, testes, ovaries, adrenal glands, brain are weighed.

Statistical analysis is performed using the Statistica program (developed by Dell Corp. Ltd., ver. 7.0). The significance of differences between data groups is assessed using Student's t-test. Differences are considered significant at p <0.05.

A macroscopic examination of the clear effect of the drug on the state of the internal organs was not established, the differences between the control and experimental groups were not found.

The body weight of the rats during the experiment is not significantly different between the experimental and control groups (table 2).

In female rats with the introduction of the drug at a dose of 400 mg / kg, the relative weight of the liver and brain in relation to the control animals significantly increased. The remaining indicators in the groups of females and males did not significantly differ from those in the control groups.

B) Assessment of acute toxicity during irrigation of the oral cavity.

The characteristics of the animals and the groups formed from them and the conditions of the study (the mode of keeping animals, the duration of the study, the estimated clinical signs, the lists of organs subjected to macroscopic examination and weighing) are similar to those specified in section A. The oral cavity is irrigated with a spray. Rats produce 2 sprays 3 times during the day, which corresponds to the doses: males 220-260 mg / kg, females 300-370 mg / kg.

The body weight of the rats during the experiment did not significantly differ between the experimental and control groups (table 3).

A macroscopic examination and weighing of the internal organs did not reveal a clear effect of the drug on the state of the internal organs, and there was no difference between the control and experimental groups.

Example 6. Evaluation of the chronic toxicity of the drug during irrigation of the mouth

Chronic toxicity for a dose of 15.6 mg / kg (TD × 20) is estimated from the results of research on outbred rats (females and males) with an initial body weight of 150-190 g of females, 200-230 g males. Animals are divided into groups (n = 5) by random sampling, taking body weight as a criterion. The variation in the initial mass does not exceed 10%. In addition, they form similar groups of control animals of each species and sex, with which a solvent is introduced in a similar way.

Animals are kept in polycarbonate cages with steel lattice lids with feed attachments (compound feed for laboratory rodents PK-120) and water. Animals receive food and water without restriction. Temperature 18-22 ° C, relative humidity 50-65%, twelve-hour lighting cycle and tenfold change of room air volume per hour. The drug, prepared in accordance with example 3, is administered by irrigation of the mouth using a spray. Euthanasia decapitation with pre-anesthesia is performed in three series on the 15th, 29th and 43th day of the experiment. An autopsy is performed on the same day.

The following indicators are determined: body weight, feed and water consumption, body temperature (rectal), heart rate. A blood test is performed to determine: 1) the number of red blood cells; 2) hematocrit; 3) platelet count; 4) the amount of hemoglobin; 5) the number of leukocytes.

A biochemical blood test is performed to determine: 1) aspartate aminotransferase; 2) alanine aminotransferase; 3) alkaline phosphatase; 4) total protein; 5) urea; 6) creatinine; 7) glucose; 8) cholesterol; 9) triglycerides; 10) total bilirubin; 11) sodium; 12) potassium.

Also perform a biochemical examination of urine and determine the following indicators: 1) leukocytes; 2) nitrites; 3) pH; 4) protein; 5) glucose; 6) urobilinogen; 7) bilirubin; 8) ketones; 9) red blood cells; 10) ascorbic acid; 11) specific weight.

Assess the behavioral reactions in the open field test (camera 100 × 100 × 60 cm, 16 squares): adaptation time (latent period), the number of crossed horizontal squares (horizontal activity), stepping on hind legs (vertical activity), washing (grooming ), acts of defecation (number of boluses), the number of urinations.

After euthanasia, the lymph nodes, the aorta, the heart, the larynx, the trachea, the lungs, the thymus, the esophagus, the stomach, the small intestine, the large intestine, the liver, the pancreas, the spleen, the kidneys, the adrenal glands, the bladder, the testes, the spleen, the kidneys, the adrenal glands, the bladder, the testes, the ovary, submandibular salivary gland with lymph nodes, the thyroid gland, the brain, the injection site (the mucous membrane of the tongue). The heart, lungs, thymus, liver, spleen, kidneys, testes, ovaries, adrenal glands, and brain are weighed.

The mass and rectal body temperature of the rats throughout the experiment did not significantly differ between the experimental and control groups (Table 4).

Gr. K. - control group

Repeated (within 28 days) irrigation of the oral cavity of rats with a medicinal product prepared in accordance with Example 3, in a twentyfold therapeutic dose, does not cause significant changes in the clinical state of the animals. Throughout the experience, no animal deaths were registered. There are no significant differences in the consumption of food and water. The drug does not cause changes in behavioral reactions, reactions to external stimuli, as well as disturbances in the motor activity of animals. The relative mass of the internal organs of rats, which are administered the drug, is not significantly different from the control.

The use of the drug does not affect the hematological blood parameters with long-term use, except that the statistical analysis of blood biochemical parameters revealed an increase in liver enzymes (ALT and ACT) in female rats, but these data do not exceed the reference parameters.

An autopsy of the animals after repeated irrigation of the oral cavity for 28 days revealed an increase in the intercellular space and a change in the shape of the cells in the liver and lungs. Changes are reversible.

Local irritation during irrigation of the oral cavity drug in the framework of the study was not identified.

Example 7. Evaluation of the allergenicity of the drug in the test in the test "Reaction of General Anaphylaxis" in Guinea Pigs

Before starting the study, the required number of animals weighing 800-850 g (females) and 1000-1100 g (males) are divided into groups by randomization by body weight. The scatter in mass does not exceed 10%. Animals are kept in cages of 5 animals each, providing free access to standard combined feed granular extruded all-in-one (GOST R 51849-2001 P.5) and water. Animals are kept in controlled environmental conditions: air temperature 20-26 ° C, relative humidity 30-70%, twelve-hour lighting cycle and tenfold change of room air volume per hour.

Sensitization is carried out in doses of TD (0.6 mg / kg) and 10 TD × 10 (6.0 mg / kg) in the form of daily inhalations of 7 minutes for 5 days. As a negative control, use 0.9% of the mass, NaCl solution. Permissive (constituting total sensitizing) doses of the drug are administered intraperitoneally 21 days after sensitization.

The clinical picture is described as follows: 0 - shock has not developed, there are no signs of it; + - mild shock (some anxiety, shortness of breath, scratching of the face, involuntary urination, defecation, wool ruffled); ++ - moderate shock (minor convulsions, pronounced effects of bronchospasm); +++ - severe shock (general convulsions, asphyxia, the animal loses its ability to hold onto its paws, falls on its side, does not die); ++++ - fatal shock.

The calculation of anaphylactic index by Weigle is carried out according to the formula:

where N is the number of guinea pigs in which death occurred;

N ₁ is the number of guinea pigs that developed severe shock;

N ₂ is the number of guinea pigs that developed mild shock;

N ₃ - the number of guinea pigs that have developed a mild shock;

N ₄ - the number of guinea pigs, which did not come shock.

With the death of all animals in the group, the Weigle index is 4 (++++), with severe shock - 3 (+++), with moderate shock - 2 (++), with weak shock - 1 (+), in the absence of anaphylactoid reactions in guinea pigs the index is 0.

Inhalation sensitization of guinea pigs at a dose of 0.6 mg / kg (TD) leads to the development of a weak anaphylactic shock in males and females of guinea pigs after the introduction of a resolving dose. The introduction of the drug in a dose of 6.0 mg / kg (TD × 10) causes a mild shock, but its manifestation is more pronounced than in the dose of TD (Table 5).

Example 8. Evaluation of drug mutagenicity in the test of micronuclei counting in mice polychromatophil erythrocytes

Animals (males) are selected and distributed into groups (n = 6), based on weight and age. The body weight of each animal does not deviate from the average for the group by more than 20%. Animals are kept in polycarbonate cages with steel lattice lids with feed attachments (compound feed for laboratory rodents PK-120) and water. Animals receive food and water without restriction. Animals are kept at a temperature of 18-22 ° C, a relative humidity of 50-65%, a twelve-hour lighting cycle and a tenfold change in the volume of room air per hour.

The drug, prepared in accordance with example 3, is administered once by spraying into the oral cavity in a volume of 0.13 ml, which corresponds to TD × 10, the preparation of research drugs is carried out 24 hours after the last injection.

As a negative control using 0.9% wt. NaCl solution (Company “DEKO” LLC, Russia), which is administered by spraying into the oral cavity four times with an interval of 24 hours. As a positive control, a solution of cyclophosphane (JSC Biochemist, Russia) is used, which is administered intraperitoneally once at a dose of 10 mg / kg 24 hours before euthanasia performed by cervical dislocation.

Preparations of bone marrow cells for recording micronuclei are prepared in a conventional manner. After euthanasia, both femoral bones are separated and cleaned of muscles. To wash out the bone marrow, fetal calf blood serum is used. 1 ml of serum is poured into the microtube. Serum is collected into a syringe and the bone marrow is washed out of both thigh bones into a microtube. Centrifuged suspension at 1000 rpm. within 5 minutes, the supernatant is removed with a plastic tip pipette, the sediment is carefully resuspended. A small drop of the suspension is placed on the edge of a glass slide, ground glass is brought to the drop at an angle of 45 ° and a smear is made, which is then dried in air. The preparations are fixed with methanol for 3 minutes, stained using the Romanovsky-Giemsa method and dried in air.

Microscopic analysis was performed by light microscopy using a LeicaDM 1000 microscope, magnification 10 × 100, oil immersion (Leica Microsystems GmbH., Germany). Analyze 2,000 polychromatophilic erythrocytes (PCE) from each animal to account for PCE with micronuclei. The proportion of PCE from the sum of PCE and normochromic erythrocytes is determined with an additional count of 400 red blood cells.

Statistical processing of results on accounting for micronuclei is carried out by comparing the experimental group with the control ones using the χ ² conjugacy criterion at a significance level for each of the comparisons of 0.05. Comparison of the proportion of PCE from the sum of all red blood cells in the control and experimental groups is carried out using the Mann-Whitney test.

ПХЭ с микроядрами, что статистически достоверно выше, чем в группах 1 и 3 (p<0,05, таблица 6).When comparing the group of mice treated with the drug (group 3) with the control group of males (group 1), there were no statistically significant differences in the frequency of PCE with micronuclei. Cyclophosphamide (positive control, group 2) induced

PCE with micronuclei, which is statistically significantly higher than in groups 1 and 3 (p <0.05, table 6).

Thus, in the test of accounting of micronuclei in polychromatophilic erythrocytes of the bone marrow of mice, it was shown that the drug prepared in accordance with Example 3 does not show cytogenetic activity after a single injection by spraying into the oral cavity, both in the highest dose recommended for humans and in doses exceeding the therapeutic 10 times.

Example 9. Evaluation of the teratogenicity of the drug

In the study, sexually mature outbred rats weighing 180–200 g were used, which were kept in separate polypropylene cages with an iron grid at an air temperature of 20–26 ° C, a relative humidity of 30–70%, a twelve-hour lighting cycle, and a tenfold change in room air volume per hour. Before starting the study, the required number of animals that meet the criteria for inclusion in the study is divided into groups by randomization by body weight with a spread in the group not exceeding 10%.

Statistical data analysis is performed using Statistica software (developed by Dell Corp., ver. 10.0). The studied parameters are averaged over the experimental groups and are presented as M ± m, where M is the group average, m is the standard deviation. The significance of differences between groups is determined using Student's t-test with p <0.05 with a normal distribution of the sample and non-parametric Mann-Whitney U-test with p <0.05 with a distribution different from normal.

The maximum daily dose of the drug (LS) "Spray 0.3% based on branched oligohexamethylene guanidine hydrochloride", prepared in accordance with example 3, for an adult person with an average weight of 70 kg in the form of sprays in the oral cavity is 3 ml (0.04 ml / kg), which corresponds to a therapeutic dose of OGMG-GC (TD) 0.5 ml (0.75 mg / kg) and a dose of TD × 10 equal to 7.5 mg / kg for a rat weighing 200 g

The drug (LS) is administered intragastrically to mature females for 15 days (3 estrous cycles) before mating in doses of TD and TD × 10. Each group consists of 10 animals. The control group females are injected with water for injection according to the same experimental design. At the end of the introduction of drugs, females are placed in intact males in a 2: 1 ratio.

Observe the general physical condition, the behavior of animals, the course of pregnancy and childbirth in female rats. Childbirth rats passed without complications. The birth date, duration of gestation, litter size, and the number of live and stillborn fetuses are recorded. In the course of the study, no deaths of rats among offspring were recorded. The results of assessment of the teratogenic effect of drugs on the reproductive function of females, recorded according to the physical parameters of the development of offspring, are presented in Table 7. The size of the litter and all indicators of rats in the experimental groups do not differ from the control indicators.

In the study of reproductive function, male drugs are administered intragastrically in doses of TD and TD × 10 within 48 days. Each group consists of 10 animals. The control group of animals according to the same scheme, water is injected. After the introduction to the males, intact females were planted in a ratio of 2: 1.

The results of the assessment of the teratogenic effect of drugs on the reproductive function of males, recorded according to the physical parameters of the development of offspring, are presented in Table 8, from which it follows that the physiological development of the offspring obtained from the experimental males is within the normal range, and the recorded indicators have no statistically significant differences from the control .

Example 10. Evaluation of the specific activity of the drug

A) In vitro studies

In the study, Streptococcus pneumoniae, Streptococcus pyogenes, Moraxella catarrhalis, Neisseria gonorrhoeae, Staphylococcus aureus, Candida parapsilosis, Candida glabrata, Candida albicans are used as test cultures. Microbial cultures are stored at -75 ° C in trypticase soy broth supplemented with 10-15% glycerol. Before the start of the experiment, bacterial strains were activated by plating on the agar medium GRM-1 (FBUN SSC PMS, Russia), incubated at 35 2 ° C for 18-20 hours.

Upon receipt of working solutions, all manipulations are performed under aseptic conditions with sterile materials. Prepare a basic solution containing 1000 μg / ml of OGMG-GC, and dilute it in nutrient broth: 2.56 ml of the basic solution is adjusted with a nutrient medium to a volume of 10 ml, and a working solution is obtained containing 256 μg / ml of OGMG-GC.

Comparative drug Tavanic®, solution for infusions, 5 mg / ml of levofloxacin (Sanofi-Aventis Deutschland GmbH, Germany, 1.0 ml) is adjusted to 5 ml with sterile distilled water in a measuring cylinder, obtaining a basic solution containing 1000 μg / ml of levofloxacin. The basic solution is diluted in nutrient broth: 2.56 ml of the basic solution is brought up to 10 ml with a nutrient medium. Get a working solution containing 256 μg / ml of levofloxacin.

Establishing the minimum inhibitory concentration (IPC) of the drug prepared in accordance with Example 1 is carried out by micromethod serial dilutions in broth in 96-well immunoassay plates.

For test cultures of microorganisms, double-dilution series are prepared in Mueller-Hinton Broth I (Oxoid) medium in a volume of 50.0 μl, sodium chloride is added to the nutrient broth to a concentration of 2%. For the preparation of the inoculum using daily culture, activated after cryopreservation. Separate morphologically homogeneous colonies are suspended in sterile 0.9% by weight. sodium chloride solution to a turbidity equivalent to 0.5 according to the McFarland standard, determined using a DEN-1 densitometer (BioSan, Latvia), which corresponds to 1.5 × 10 ⁸ CFU / ml. Subsequent dilution of the suspension is prepared by diluting to a titer of 2 × 10 ⁶ CFU / ml.

50 μl of the inoculum is applied to each cell containing 50 μl of drug at a concentration of 256 μg / ml; the final concentration of the drug is 128 μg / ml. To control the growth of 3 cells, 50 μl of nutrient broth without drugs (“growth control”) are added. As a “negative control,” dilutions of the substance and monk were added to the wells, which were stored at 4 ° C until the results were recorded. The final titer of microorganisms in each cell is 1 × 10 ⁶ CFU / ml. The inoculum is introduced into the cells with dilutions of drugs not later than 30 minutes from the moment of its preparation. The range of concentrations of OGMG-GC is from 128 to 0.001 µg / ml.

The plates with the tested strains are incubated in ordinary atmosphere at a temperature of 35 ± 2 ° C for 20-24 hours. To determine the presence of growth plates with crops see in the transmitted light. The growth of cultures in the presence of drugs is compared with the negative control containing the original inoculum and stored at 4 ° C. BMD is determined by the lowest drug concentration, which inhibits the visible growth of microorganisms.

According to the results of the experiment, it was established that the studied microorganisms are not sensitive to drugs prepared in accordance with Example 1. The most pronounced activity of drugs is in relation to Streptococcus pneumoniae and Streptococcus pyogenes (BMD 0.002-0.25 μg / ml), Moraxella catarrhalis (BMD 0.03-0.06 μg / ml), Neisseria gonorrhoeae (MPK 0.125-2 μg / ml ). Somewhat less stable activity was observed for Staphylococcus aureus (methicillin-sensitive and methicillin-resistant, MIC 1-2 µg / ml). Candida parapsilosis and Candida glabrata cultures (IPC 32-64 µg / ml) showed low sensitivity. In relation to Candida albicans, the drug is more active (MIC 2 µg / ml).

B) In vivo studies on the model of traumatic stomatitis and gingivitis

Before starting the study, the required number of animals that meet the criteria for inclusion in the study is divided into groups by randomization by body weight (3.2-3.5 kg). The spread does not exceed 10%. Animals are kept in individual cages under controlled environmental conditions: air temperature of 16-22 ° C, relative humidity of 30-70%, a twelve-hour lighting cycle and a tenfold change in room air volume per hour. Rabbits are provided with free access to standard feed pelleted all-extruded (GOST R 51849-2001 P.5) and water.

Oral irrigation of experimental animals is carried out 3 times a day with 2 sprays, which is 0.5-0.6 ml of solution (for a 0.1% solution of approximately 0.2 mg / kg, for a 0.3% solution of approximately 0.6 mg / kg). The solutions of the test drug is applied by spraying using a plastic spray dispenser (40 ml) for 7 days. Solutions are prepared daily immediately before use.

Traumatic stomatitis and gingivitis are reproduced on 18 rabbits of the Chinchilla breed (3 males and 3 females in the group) under anesthesia (0.7 ml Zoletil 100 + 1.3 ml Rometar) by thermal burn of the oral mucosa in the region of the lower incisors and cheeks with red-hot red a plugger with a diameter of 3 mm for 7-10 seconds.

After 24 hours, begin treatment with 0.1% and 0.3% solutions of the tested drug. In the control group, sterile water for injection is sprayed in a similar way as with the experimental groups. The effect of the substance was judged by the rate of wound healing (visually) and the pathological conclusion.

Clinical observation of animals in cages is carried out daily, and deviations in the consumption of feed and water of animals in individual cages are also visually noted. After treatment, animals of all experimental groups are euthanized, followed by taking samples of the affected areas of the oral mucosa for histological studies in order to compare the effect.

The study of specific activity shows that during the experiment, the dynamics of the body mass of animals remains positive, their physiological state, appetite and water consumption are normal. After 7 days, the majority of animals in the experimental groups (0.1 and 0.3% DV) showed a decrease in mucosal edema, a decrease in tissue hyperemia, and a pink-red mucosa was observed under partially removed fibrinous plaque.

Microscopic examination of all histological preparations of the mucous membranes of the cheeks of the maxillary zone and gums of the peridental areas of the mandibular zone obtained from rabbits after topical application of a solution of drugs at concentrations of 0.1 and 0.3% reveal similar manifestations of pathological changes that characterize a weak inflammatory process and are pronounced regeneration of damaged tissue. The mucous membrane of the maxillary zone of the cheek is covered with stratified squamous epithelium with areas of keratinization. On the surface of the mucous membrane, some minor erosive-ulcerative damage is observed, as well as moderate proliferation of granulation tissue and almost complete restoration of the epithelium lining the mucosa. The buccal muscles and salivary glands of the cheek are intact.

The mucous membrane of the gums of the peridental areas of the mandibular zone of the experimental animals is covered with a stratified squamous keratinized epithelium. On the surface of the mucous membrane, spreading to the area of the holes of the lower incisors, erosive and ulcerative damage is found at the healing stage with the development of pronounced regeneration. Inflammatory infiltration and hemorrhages are not observed.

The mucous membrane of the maxillary cheek area and the gingival membrane of the peridental areas of the mandibular zone in the control group are characterized by the presence of focal erosive-ulcerative lesions and weak proliferation of granulation tissue, areas of inflammatory infiltration and hemorrhage are noted.

Thus, in the study of the mucous membranes of the oral cavity of rabbits, it is concluded that the defects in the application groups of the studied drug are almost completely healed at a concentration of 0.1% and 0.3% 7 days after the thermal formation of the model of stomatitis and gingivitis.

In accordance with the instructions for use of the spray “Teraflu® LAR, 0.2%”, the maximum daily dose for an adult (4 sprays 6 times a day) is 3 ml of the solution, which contain 6 mg of the active substance (BOX). With an average body weight of 70 kg, the dose is expressed as 0.086 mg / kg. The equivalent daily maximum dose for a rabbit, taking into account the metabolic coefficient of 3.2, is 0.28 mg / kg.

Based on the data obtained, the cure of traumatic stomatitis and gingivitis in a rabbit with an average body weight of 3.5 kg is achieved by spraying into the oral cavity a 0.6 ml drug solution containing 0.1% w / v. OGMG-GC as the main active substance, which corresponds to a dose of 0.17 mg / kg, which is 1.65 times lower than for BOC in the case of the spray “Teraflu® LAR, 0.2%”. This indicates a higher efficacy of the medicinal product prepared in accordance with the invention compared with the drug present on the market.

Claims (11)

1. Drug in the form of a solution to obtain a spray for the treatment of diseases of the oral cavity, containing a combination of two active ingredients, excipients and a solvent, characterized in that the first active ingredient is a branched oligohexamethyleneguanidine of the formula

, where R is

n ₁ , n ₂ and n ₃ are 1-3, az is 0.15-1.10 with a molecular mass distribution of M _w / M _n from 5.4 to 9.3 with a mass-average molecular weight M _w in the range from approximately 3800 to 6300 and the number average molecular weight of M _{n is} in the range of from about 600 to 1100 in the form of hydrochloride (OGMG-GH), and the second active ingredient is selected from benzalkonium chloride

or benzoxonia chloride

excipients include glycerin, menthol, sodium saccharinate, and the solvent is purified water or its mixture with ethyl alcohol, while the drug has a composition, wt./vol.%: OGMG-GH                             0.1-0.5 benzalkonium chloride           0.005-0.05 or benzoxonia chloride 0.01-0.02 glycerol                               30-50 menthol                         0.01-0.03 sodium saccharinate             0.03-0.06 solvent                             up to 100 2. Drug under item 1, characterized in that it has a composition, wt./vol.%: OGMG-GH                     0.1-0.3 benzalkonium chloride   0.005-0.05 glycerol                       30-40 menthol               0.01-0.025 sodium saccharinate     0.03-0.05 solvent                     up to 100