WO2017003403A1

WO2017003403A1 - The use of decamethoxin as a pharmaceutically active substance for orally treating gastrointestinal diseases and intestinal infections

Info

Publication number: WO2017003403A1
Application number: PCT/UA2016/000079
Authority: WO
Inventors: Наталия Мыколаивна ДЕРКАЧ
Original assignee: Наталия Мыколаивна ДЕРКАЧ
Priority date: 2015-07-02
Filing date: 2016-07-02
Publication date: 2017-01-05
Also published as: EA201890181A1; EA035006B1

Abstract

The invention relates to the oral use of decamethoxin as a pharmaceutically active substance for treating gastrointestinal diseases, as well as intestinal infections with microbial and viral etiology.

Description

APPLICATION OF DECAMETOXIN AS A PHARMACEUTICALLY ACTIVE SUBSTANCE FOR THE TREATMENT OF DISEASES OF THE GASTROINTESTINAL

TRACT AND INTESTINAL INFECTIONS ORAL

The technical solution relates to the new use of decamethoxin, namely, to the use as a pharmaceutically active substance for the treatment of diseases of the gastrointestinal tract, as well as intestinal infections of microbial and viral etiology.

State of the art

Decamethoxin is a chemical compound [1, 10-decamethylene bis (N, N - dimethylmentoxycarbonylmethyl) ammonium dichloride], a white crystalline powder with a faint odor, readily soluble in water, close to ethonium in chemical structure and antimicrobial effect. It has a wide range of antimicrobial effects on gram-positive (staphylococcus, streptococcus, pneumococcus), gram-negative (gonococcus, meningococcus) cocci, corynebacteria diphtheria, enterobacteria, pseudomonas, protozoa, dermatophytes, yeast-like fungi of the genus Candida. In the process of application, microorganism variants resistant to decamethoxin are formed very slowly. (http://compendium.com.ua/akt/68/610/decametoxinum).

Due to the fact that decamethoxin is a surfactant, it changes the permeability of the membrane of the microbial cell, leading to the destruction and death of microorganisms, which determines its bactericidal effect [Pashynskaya V.A., 2002; Shchetina V.N., 1990]. The antiseptic effect of the substance is based not only on bactericidal, but also on bacteriostatic activity. The bacteriostatic effect is due to the following mechanisms: a) Inactivation of exotoxins of microorganisms, which was demonstrated on pathogens of diphtheria, and which made it possible to recommend decamethoxin in 1994 to combat the epidemic of diphtheria [Kovalchuk VP, 2002];

b) Decrease in adhesion of pathogenic microorganisms [Zhornyak O.I., Stukan OK, Sukhlyak VV, 2010; Zhornyak O.I., Stukan O.K., 2010]. For example, decamethoxin at a concentration of 10 μg / ml dramatically reduces the adhesion of corynebacteria, salmonella, staphylococci and Escherichia. A decrease in adhesion can be achieved either by competitive binding to bacterial adhesins or to surface receptors of the host cells.

c) Destruction of fimbria proteins, flagella located on the membrane surface (as you know, the main function of fimbria is to ensure adhesion of gram-negative bacteria to the host cells, and flagella - the movement of microorganisms).

d) Blocking the function of the cell wall (transmembrane transport of metabolites, nutrition, respiration), inhibition of cellular respiration, protein synthesis and cell division. So, it was shown that decamethoxin

one

FIXED SHEET (RULE 91) ISAUA blocks the respiratory chain of Pseudomonas aeruginosa [Bievski ĭ AN, 1994]; that in the presence of decamethoxin, the activity of dehydrogenases in Escherichia coli, Pseudomonas aeruginosa, Micrococcus lysodeikticus [Shchetina VN, 1990], staphylococcus dehydrogenase and Candida albicans [Moroz V.M., 2002], which indicates inhibition of aerobic oxidation of glucose, succinate and pyru, is significantly reduced while the activity of aspartate and alanine aminotransferase, on the contrary, is increasing [Shchetina VN, 1990]; forms complexes with bacterial DNA [Sorokin VA, 1990; Kukhar VP, 1989]. Violation of cell metabolism and division provides a significant therapeutic effect without lysis of the microbial cell [Pasechnik SP.; Feshchenko Yu. 1., 2002].

Decamethoxin is used for topical treatment of diseases of the oral cavity (stomatitis, ulcerative necrotic gingivitis, degenerative-inflammatory form of periodontosis of I-II degree in the acute stage), pharynx, larynx (with gingivitis, candidiasis of the oral mucosa, pharyngitis, laryngitis, tonsillitis); for sanitation of the oral cavity, pharynx, nasopharynx in carriers of pathogenic staphylococcus, diphtheria bacillus, candida; prevention of secondary infection after surgical interventions in the oral cavity, pharynx, larynx; locally also for the treatment of pustular bacterial and fungal skin diseases, microbial eczema, purulent-inflammatory lesions of the soft tissues (abscesses, carbuncles, phlegmon, boils, purulent wounds, panaritiums) .Also used endobronchially for lung abscesses, bronchiectasis diseases, cystic hypoplasia suppuration, chronic bronchitis in the acute phase. Topically applied for proctitis and ulcerative colitis. In ophthalmology in the form of eye drops, it is used for conjunctivitis and blepharoconjunctivitis and for the treatment of contact lenses. Decamethoxin is also used to sterilize medical instruments, devices, suture material, rubber gloves and for chemical sterilization and preservation of bone-tendon grafts [http://uk.wikipedia.org/].

Nifuroxazide - an intestinal antiseptic, a derivative of 5-nitrofuran; active against most pathogens of intestinal infections (including mutant strains resistant to other antimicrobial agents). It exhibits a local antibacterial effect in the enlightenment of the intestine regarding certain species of gram-positive bacteria from the family Staphylococcus and some species of gram-negative bacteria from the family Enterobacteriaceae: species Yersinia spp., Escherichia spp., Citrobacter spp., Enterobacter spp., Klebsiella spp., Salmonella spp. The disadvantage of the tool is that, unlike decamethoxin, it does not show an antibacterial effect on bacteria of the species Proteus vulgaris, Proteus mirabilis and Pseudomonas aeruginosa

[http://compendium.com.ua/info/172054/gedeon-richter/nifuroksazid-rikhter].

Phthalazole, like other sulfonamide drugs, prevents the inclusion of PABA in the synthesis of follic acid in microbial cells. it leads to a violation of the formation of folic acid, which is involved in the synthesis of purine and pyrimidine bases, on which the growth and development of microorganisms depends. This effect develops gradually, because the microbial cell has some reserves of PABA. Therefore, phthalazole 5 should be administered in sufficiently high doses. Otherwise, persistent strains of pathogens can form, which are not amenable to further action of sulfa drugs. It has a contraindication: increased individual sensitivity to sulfa drugs; blood system diseases; diffuse toxic

Yu goiter; chronic renal failure; glomerulonephritis; acute hepatitis; intestinal obstruction. And it also has a number of indirect actions from the side: the nervous system, cardiovascular system, blood system, gastrointestinal tract, respiratory system, urinary system, allergic and toxic-allergic reactions

₁₅ [http://www.rlsnet.ru/tnjndex_id_5378.htm].

Furazolidone is an antibacterial drug of the nitrofuran group, a synthetic derivative of 5-nitrofurfural, which has a pronounced antimicrobial activity against gram-negative aerobic microorganisms, smaller measure Q is gram-positive aerobic microorganisms sensitive to the action of the drug, some of the simplest and fungi (in particular, fungi of the genus Candida). The pharmacological effect of the drug directly depends on the dose, when using low doses, furazolidone provides a bacteriostatic effect, with an increase in the dose, pronounced bactericidal activity is noted. The disadvantages of this tool is that it cannot be taken for a long time, because with prolonged use of the drug, the possible development of hemolytic anemia and methemoglobinemia (mainly in newborns and infants), as well as shortness of breath, cough, hyperthermia and neurotoxic reactions. When using high doses of the drug in patients, the possible development of 0 toxic liver damage, including acute toxic hepatitis.

In addition, the possible development of polyneuritis (neurotoxic effect) and the hematotoxic effects of furazolidone

[http://www.piluli.kharkov.ua/drugs/drug/furazolidonum/].

Intestopan - the drug has antibacterial and antiprotozoal 5 (suppresses the vital activity of the simplest organisms) activity and is close to the mechanism of action of chlorchinaldol. The disadvantage of this tool is the presence of bromine in the molecule of the active substance, which can lead to dangerous indirect effects on the body [http://www.piluli.kharkov.ua/drugs/drug/2211/].

0 Despite the wide range of effects of the agents indicated in the prior art for the treatment of intestinal infections of microbial and viral etiology, namely: salmonellosis, dysentery, diphtheria, enteritis, gastritis, gastroenteritis, enterocolitis, gastroenterocolitis, brucellosis, proctitis, sigmoiditis, colitis, etc. , there are some diseases that 5 can only be treated surgically, for example, infectious coloproctitis.

FIXED SHEET (RULE 91) ISAUA Infectious coloproctitis is an inflammation of the tissues that surround the rectum.

For various reasons, and sometimes without an existing reason, a micro-hole is formed in the anal canal, through which the infection from the rectal lumen penetrates into the peri rectal tissue. Inflammation develops, an abscess forms, which inevitably opens out or into the lumen of the rectum.

After the usual opening of the abscess in acute infectious coloproctitis, without eliminating its internal opening, rectal fistulas (chronic infectious coloproctitis) are formed in 80-98 %% of patients.

Acute infectious coloproctitis usually begins acutely, the clinical picture is quite bright: there is a deterioration in general position, weakness, malaise, fever, chills, pain in the anus and perineum. In the area of the anus, a small painful swelling forms, from which later an abscess develops, which can be located directly under the skin of the perineum, and deep between the muscles of the perineum and buttocks.

If adequate treatment is not timely performed, inflammation spreads to adjacent cellular spaces, an abscess breaks into the rectum, or out through the skin of the perineum. The pain subsides, the body temperature decreases, the general situation of the patient improves. But an abscess breakthrough does not lead to a complete recovery, the disease continues to progress with the formation of new rectal fistulas (chronic infectious coloproctitis).

Treatment of acute infectious coloproctitis is only surgical. All patients, without exception, need emergency surgical care.

The basic principles of the operation:

1) opening and drainage of the abscess;

2) the elimination of its internal opening through which the abscess cavity communicates with the rectum.

The operation is performed under general anesthesia.

After the operation, dressings are performed, antibiotics, vitamins, and drugs that increase immunity are prescribed.

Opening the abscess is not a radical operation: after it, as a rule, repeated suppuration occurs (chronic infectious coloproctitis forms). The reason for this phenomenon is the preservation of the incendiary channel (fistula) between the rectum and surrounding tissues. In this case, re-infection of the intestinal flora through the internal opening of the fistula, which constantly supports the inflammatory process, occurs.

For complete treatment, a second operation is necessary - radical, therefore it leads to complete cure. As a result of such an operation, the connection between the intestinal cavity and the abscess is eliminated.

FIXED SHEET (RULE 91) ISAUA The prolonged existence of an incendiary focus in the perineum leads to the appearance of irritability, insomnia, headache, decreased performance, the possible development of neurasthenia, impotence. Procrastination worsens not only the general position of the patient, but also the prognosis, since the likelihood of a purulent process spreading through the cellular spaces of the pelvis, destruction of the muscular structures of the sphincter, pelvic floor and rectum wall increases.

To date, the only method of treating patients with rectal fistulas is a radical operation, when, in addition to opening the abscess, the internal opening is eliminated, which has become the source of this disease [http://www.doctor-maximov.ru/paraproktit/].

As noted above, treatment of acute infectious coloproctitis is possible only surgically, during the operation only mechanical elimination of the abscess and source of inflammation is performed, drainage of pus is performed with antiseptic agents. But this only leads to a partial recovery of the patient, because the microbes that are in the enlightenment of the gastrointestinal tract can easily get to the already damaged area, which is just starting to heal, and this can lead to the resumption of the inflammatory process and the transition of the acute form of the disease to chronic.

It is in order to avoid the entry of bacteria from other parts of the gastrointestinal tract, it is advisable to use agents that have antimicrobial properties not only as a local agent, but also as an oral agent that will destroy pathogenic microflora in enlightening all sections of the gastrointestinal tract, which can provoke a relapse of the disease. The advantage of the oral administration of a pharmaceutically active substance is that its effect extends to all parts of the gastrointestinal tract, because it envelops all the mucous membranes when the substance passes from the oral cavity to the rectum, and not some specific area, as when using locally acting agents. It should also be noted that when using local remedies, a person needs the help of a specialist or special means for carrying out manipulation, which, of course, complicates the treatment of the disease. In this case, the oral application has a very significant advantage, because it does not need outside help and can be carried out by patients independently and even at home.

An analysis of publications on the use of decamethoxin showed that it was never used as a means for oral administration. Known methods for using this antiseptic were limited only by the short-term action of decamethoxin on the surface of the skin or mucous membranes. These methods include: inhalation, rinsing, sanitation, drainage with decamethoxin solutions, lotions, applying ointments, gaels, pastes, which include decamethoxin as one of the components.

The reason that decamethoxin was not used as

FIXED SHEET (RULE 91) ISAUA oral medication may be the lack of information on acute toxicity of decamethoxin, chronic and specific toxicity with prolonged enteral administration of decamethoxin, possible local irritating and allergenic effects, its absorption and effect on blood cholinesterase activity, the effectiveness of decamethoxin as an antimicrobial agent in vivo is also uncertain the reason for not using decamethoxin is its belonging to surfactants. Thus, there is a strong belief about the use of decamethoxin exclusively as a surfactant, that is, for surface treatment, not for internal application. Another reason for not using decamethoxin as an internal remedy is its very bitter and unpleasant taste.

Disclosure of invention

The objective of the technical solution is to improve the treatment of diseases of the gastrointestinal tract and intestinal infections, in particular, microbial and viral etiology. Another objective of this technical solution is to expand the range of pharmaceutically active substances for the treatment of diseases of the gastrointestinal tract and intestinal infections. Another objective of the presented technical solution is to expand the range of pharmaceutically active substances for the treatment of infectious coloproctitis without surgical intervention.

The problem is solved by the use of decamethoxin as a pharmaceutically active substance for the treatment of diseases of the gastrointestinal tract and intestinal infections orally.

In addition, decamethoxin can be used to treat diseases of the gastrointestinal tract and intestinal infections of microbial and viral etiology.

In addition, decamethoxin can be used to treat infectious coloproctitis.

In addition, decamethoxin can be used in the form of an aqueous solution with a concentration of decamethoxin 0.02-0.2 mass%.

In addition, decamethoxin can be used as part of a pharmaceutical composition.

In addition, decamethoxin can be used in the pharmaceutical composition of the liquid type.

The authors of the technical solution proposed a new application of decamethoxin - the use for the treatment of diseases of the gastrointestinal tract and intestinal infections, based on the antimicrobial effect shown by it on a wide range of microbes that cause gastrointestinal disease. For this, studies were conducted regarding its toxicity when administered orally, and regarding the study of its effect on a number of microorganisms that cause gastrointestinal tract disease. As a result, we studied the new properties of decamethoxin and its effect on diseases that were not previously subject to medical treatment, and the proven possibility of its use

FIXED SHEET (RULE 91) ISAUA orally due to the lack of toxic effects on the body.

Brief Description of the Drawings

To determine the effectiveness and carelessness of using decamethoxin orally for the treatment of diseases of the gastrointestinal tract, studies were conducted, the results of which are presented further in the form of diagrams, graphs and tables. Schemes and graphs are shown in figures 1-4.

FIG. 1 is a diagram of a method for determining the anticholinesterase activity of inhibitors for unspent acetylcholine using an indicator reaction of the combined oxidizing p-phenethidine (chromogen) with hydrogen peroxide, in which p-phenethidine is used as an indicator reaction to oxidize p-phenethidine with peracetic acid formed in the initial perhydrolysis reaction (with hydrogen peroxide), which allows you to control the activity of the cholinesterase enzyme in the presence of an exogenous inhibitor - decamethoxin (Inhibit p).

FIG. 2 is a graph with kinetic curves of the combined oxidation of p-phenethidine with hydrogen peroxide in the presence of acetylcholine (hereinafter abbreviated AX) - this is a control sample, AX + cholinesterase (hereinafter abbreviated XE) is a single sample, mixtures (XE + decamethoxin) + AX is a calibration test, mixtures (XE + perfusate) + AH - this is the test sample.

FIG. 3 is a graph with kinetic curves of the combined oxidation of p-phenethidine with hydrogen peroxide in the presence of AH - this is a control sample), a mixture of AH and endogenous CE of the blood plasma of rabbits of the control group k, 30 and 60 times after the administration of 0.9 %% sodium chloride solution.

FIG. 4 is a graph with kinetic curves of the combined oxidation of p-phenethidine with hydrogen peroxide in the presence of AH. This is a control sample of a mixture of AH and endogenous CE of the blood plasma of experimental rabbits group k, 30 and 60 times after the administration of the decamethoxin solution.

Option (s) for carrying out the invention

To determine the carelessness of using decamethoxin orally for the treatment of diseases of the gastrointestinal tract (the gastrointestinal tract is further reduced), the authors conducted the following studies:

1. Study of the ability of decamethoxin to be absorbed from the gastrointestinal tract (resorptive effect) by determining its concentration in the intestinal perfusate in situ and the effect on blood XE activity upon oral administration.

The experiments were performed on 5 white randomized male rats and 8 chinchilla rabbits.

The isolated section of the intestine was perfused with a solution of decamethoxin at a concentration of 0.02-0.2 %% for 30 minutes. The content of decamethoxin was determined in perfusion by the reaction of inhibition of exogenous CE. The degree of inhibition of the reaction in the presence of perfusate and a solution of decamethoxin with a concentration of 0.02 mass %% was compared, which made it possible to determine the content of the latter in the perfusate. Decrease in concentration

, 7

FIXED SHEET (RULE 91) ISAUA decamethoxin in perfusate compared with that in the initial solution indicates the absorption of this compound in the intestines of rats.

In the experimental group of rabbits, the ability of a decamethoxin solution to be absorbed into the blood was determined due to the effect on CE activity 30 and 60 minutes after a single oral administration at a dose of 6 ml / kg, which corresponds to 1.2 mg / kg of decamethoxin. Blood samples were taken to, 30 and 60 minutes after the introduction of a solution of decamethoxin. The control group animals were injected with 0.9 %% sodium chloride solution instead of decamethoxin solution.

As an indicator reaction to acetylcholine, we used the oxidation of p-phenethidine with peracetic acid (CH3CO3H) formed in situ in the auxiliary perhydrolysis reaction (with excess hydrogen peroxide) of acetylcholine, a substrate of the enzymatic reaction. A diagram of this method is shown in FIG. 1. A solution of CE 200 AO / ml (horse serum acylhydrolase K.F.3.1.1.8 NPO Biomed, Russia VI cells, 80 mg vial with an activity of 25 AO / mg) and a substrate solution of acetylcholine were used. The determinations were performed spectrophotometrically at 358 nm in a cuvette with a layer thickness of 10 mm. The rate of this reaction characterizes the slope of the rectilinear initial portion of the kinetic curve after the induction period in the coordinates "optical density of the product of the oxidation of p-phenethidine - hour".

The activity of CE in the blood of rabbits was determined by the enzyme-kinetic method described above. A decrease in CE activity 30 and 60 minutes after administration of decamethoxin solution to animals in comparison with the initial and in animals of the control group served as a marker of decamethoxin absorption in the gastrointestinal tract.

The results of the study are presented in figures 2-4 and in tables 1-3. The value of the slope of the test samples and the results of quantitative determination of the contents of decamethoxin in rat intestinal perfusate are presented in table 1. The difference in the values of the slope of the slope of the control and blank samples is a range that reflects the relative change in CE activity from the conditional 100 %% (blank sample) up to 0 %% (control sample).

The content of decamethoxin in the solution is 0.200 mg / ml, in the perfusate - 0.195 ± 0.003 mg / ml. There are no statistically significant differences between the decamethoxin content in the initial solution and rat intestinal perfusate. This suggests that decamethoxin is not absorbed in the intestines.

Table 2 shows the experimentally obtained values of the slope of the kinetic curves of the samples and the change in the slope of the kinetic curve, which corresponds to a change in cholinesterase activity by 1%, under the influence of 0.02 mass% decamethoxin solution. The initial activity of CE determined in the blood plasma of rabbits to the introduction of a solution of decamethoxin is taken as 100%.

8

FIXED SHEET (RULE 91) ISAUA Table 1

The study of decamethoxin content in rat intestinal perfusate

table 2

The value of the slope of the kinetic curves of the samples and

changes in the slope of the kinetic curve,

which corresponds to a 1% change in cholinesterase activity

The results of determining the activity of CE are shown in table 3.

Table 3

The change in rabbit plasma cholinesterase activity in the experiment under the influence of 0.02 mass% decamethoxin solution

There were no statistically significant differences in the activity of the plasma enzyme of rabbits after administration of a decamethoxin solution between the control and experimental groups. This indicates the absence of absorption of decamethoxin from the gastrointestinal tract of rabbits, which confirms the above data on the absence of its absorption from the small intestine of rats. The research results regarding the effect of a decamethoxin solution with a concentration of 0.2 mass% had approximately the same results as for a decamethoxin solution with a concentration of 0.02 mass%, therefore, these data are not presented for economy.

Based on this, it can be concluded that the use of decamethoxin orally is safe - because it is practically not absorbed in the digestive tract, acts only in the intestinal lumen and does not affect the vital functions of other organ systems.

All studies were carried out on the basis of a solution of decamethoxin in water; for presentation in the application materials, concentrations with a variation of 0.02-0.2 mass% were selected, however, for the treatment of different degrees

10

FIXED SHEET (RULE 91) ISAUA gastrointestinal diseases and intestinal infections of microbial and viral etiology orally, other concentrations can be used, because these examples are given only to illustrate the results of studies.

2. The study of the parameters of acute toxicity of decamethoxin solution.

The acute toxicity of the decamethoxin solution after oral administration was also studied on outbred rats of both sexes (n = 60) and outbred mice of both sexes (n = 72) with the determination of the semi-legal dose (LD50). Oral administration to rats of a maximum of 0 permissible volume (5 ml) of decamethoxin solution confirmed the absence of intoxication in animals and the impossibility of establishing LD50. Determination of LD50 of decamethoxin solution in rats (the results of the study are presented in Table 4) showed that the LD50 value of decamethoxin solution is in the dose range of 500-750 mg / kg for rats, and after 5 additional studies it was 586 mg / kg with intragastric administration. In accordance with the generally accepted classification, a solution of decamethoxin for a single intragastric administration to rats belongs to class IV of low toxic substances (500 mg / kg <LBBO <5000 mg / kg). 0 table 4

Final study of acute toxicity of rat decamethoxin solution with intragastric administration

5 Determination of the effect on a functional state of an organism of a single intragastric administration of a toxic dose of a decamethoxin solution that does not cause death (400 mg / kg) was carried out on 12 animals, after which the animals were observed for 14 days.

The general position of the animals, mortality, and the dynamics of body mass Q were evaluated, and upon completion of the experiment, after the animals were removed from the experiment, a macroscopic assessment of the condition of the internal organs and systems was performed, and the mass coefficients of the internal organs and histological examination of the internal organs were calculated on days 2 and 14 after decamethoxin administration. Analyzing the obtained macro- and microscopic data, 5 it can be concluded that a single intragastric administration

eleven

FIXED SHEET (RULE 91) ISAUA a solution of decamethoxin in a dose of 400 mg / kg leads in the early stages of observation (after 2 days) to the development of signs of irritation of the gastric mucosa, a reactive shift in the thymus, some tension of adrenocorticocytes of the bundle cortex and chromafin cells of the adrenal medulla, a slight decrease in the protective capabilities of the mucous membrane small intestine. These changes are temporary and 14 days after the intragastric administration of a solution of decamethoxin at a dose of 400 mg / kg, morphological abnormalities in the above tissue structures are not recorded. On the part of other organs and systems, no visible changes were found in any of the study periods. The results of the study are presented in tables 5-6.

Table 5

Dynamics of body weight of rats of both sexes (g) after a single intragastric administration of a toxic dose of decamethoxin solution

Oral administration to rats of the maximum allowable volume of decamethoxin (0.02 mass% solution) confirmed the absence of intoxication in animals. The LD50 of a decamethoxin solution (586 mg / kg) makes it possible to classify decamethoxin as a class IV low toxic compound (500 mg / kg <LD50 <5000 mg / kg). The revealed morphological changes with the introduction of a solution of decamethoxin at a dose of 400 mg / kg (the maximum toxic dose that does not cause the death of animals) are temporary and are not recorded after 14 days. A study regarding the effect of a decamethoxin solution with a concentration of 0.2 mass% showed its LD50 of 734 mg / kg - which makes it possible to classify decamethoxin as a class IV low toxic compound (500 mg / kg <LD50 <5000 mg / kg), in addition, morphological changes were revealed when the introduction of a solution of decamethoxin in a dose of 400 mg / kg (the maximum toxic dose that does not cause the death of animals) is temporary and after 14 days are not recorded.

The data obtained indicate that decamethoxin in the form of 0.02 mass% solution and 0.2 mass% solution when administered orally is relatively safe. Table 6

Ratios of rat internal organs mass (g / 100 g) of both sexes after a single intragastric administration of a toxic dose

decamethoxin solution

Studies have been conducted on the possible manifestations of the locally irritating, allergenic action of decamethoxin. Studies of the local irritating effect with subconjunctive administration were carried out on chinchilla rabbits (n = 9) and when administered orally on outbred rats of both sexes (n = 48) with the following pathological examination of the mucous membrane of the stomach and intestines. Decamethoxin solutions of 0.02 mass% and 0.2 mass%, in a volume of 0.01 ml, were introduced into the conjunctival sac of the rabbit's right eye. The left eye served as a control; distilled water was injected in a similar volume. The research results are presented in table 7 and table 8 (note: 0 - there is no reaction to animals; n = 3 is the number of animals).

Thus, it was determined that 0.02 mass% and 0.2 mass% solutions of decamethoxin, when applied locally in a therapeutic dose, do not give a locally irritating effect on the mucous membranes of laboratory animals.

Studies of the locally irritating effect of decamethoxin (0.02 mass% and 0.2 mass% solutions) when administered orally were studied as part of a chronic toxicity study and were performed on rats that were administered intracranial decamethoxin at doses of 3 ml / kg and 30 ml / kg for 28 days daily. Daily mortality, behavior, activity, food and water intake were assessed. Table 7

Results of a study of the effect of decamethoxin solution on

The eye condition in rabbits with topical application, n = 3

Continuation of table 7

Table 8

The results of a study of the effect of decamethoxin solution on the eye condition in rabbits with local application, n = 3

Continuation of table 8

Morphological studies of the esophagus, stomach, small, large and rectum after 28 days showed the absence of any changes in the gastrointestinal tract, which confirms the absence of the irritating effect of 0.02 mass% and 0.2 mass% of decamethoxin solutions on the listed organs of rats.

Output. Thus, a preparation in the form of 0.02-0.2 mass %% decamethoxia solution with long-term (28 days) oral administration does not create a locally irritating effect on the gastrointestinal mucosa of laboratory animals in a therapeutic dose.

3. Studies of the allergenic effect of decamethoxin were performed using the oral route of administration in guinea pigs (n = 60) and the parenteral route of administration in mice (n = 40) using the tests “active skin anaphylaxis”, “conjunctive test”, “hypersensitivity” slow type. "

a) Test decamethoxin model of active skin anaphylaxis

(ASA) in guinea pigs (n = 30) showed no reaction (see table 9). The ASA model is a reaction of skin capillaries at the site of intradermal administration of an antigen (in this case, 0.02 mass% and 0.2 mass% solutions of decamethoxin with actively sensitized guinea pigs. Intravenous administration of a dye solution that is easily spread by blood throughout the body, and due to leaving the bloodstream at the site of tissue inflammation (intradermal administration of the antigen) forms a blue spot.The absence of a reaction showed that decamethoxin is not a potential allergen, since when administered orally at a dose 3 ml / kg and 30 ml / kg does not cause sensitization of the body and the development of an anaphylactic reaction of the immediate type.

Table 9

Determination of the allergenic effect of 0.2 mass% decamethoxin solution, on the model "Active cutaneous anaphylaxis", Me (Q25; Q75)

b) The study of the allergenic effect of 0.02 mass% and 0.2 mass% of decamethoxin solutions in the test “Conjunctive test” in guinea pigs (n = 30) showed the absence of an allergic incendiary reaction of the mucous membrane of the eye (see table 10). Expressiveness ophthalmic reaction depends on the ability of the allergen (the studied drug) to sensitize the body and at the place of the final dose, sharply increase the permeability of the walls of the capillary vessels and, therefore, cause tissue edema at the site of administration of the antigen.

On the 10th, 20th and 30th day, sensitized orally animals were tested: 1 drop of the test drug was administered under the upper eyelid of animals in a concentration that did not cause local irritating effect in intact animals. The second eye served as a control. After 15 minutes, 1 hour (immediate-type hypersensitivity reaction) and 24 hours (delayed-type hypersensitivity), the severity of ophthalmic reaction was assessed on a scale (from 0 to 3 - from the lack of reaction to the redness of the entire conjunctiva). According to the data of table 10, in all animals of the experimental groups, the absence of an allergic incendiary reaction of the mucous membrane of the eye was observed. Decamethoxin solutions 0.02 mass% and 0.2 mass% in doses of 3 and 30 ml / kg do not cause sensitization of the body and the development of an allergic reaction of instant and delayed type in the test "Conjunctive test"

Table 10 Determination of the allergic effect of 0.2 mass% solution

decamethoxin in the test “Conjunctive test”, Me (Q25; Q75)

c) Study of the allergic effect of 0.02 mass %% and 0.2 mass %% solutions of decamethoxin in a delayed-type hypersensitivity reaction (GTS) in mice (n = 40) at a dose of 1, 3 mg / kg and 13 mg / kg, not sensitizes the animal organism and does not cause the development of allergic inflammation, which proceeds according to a delayed type of hypersensitivity. This reaction allows us to identify the ability of the test substance to sensitize lymphocytes-ectors, which leads to the release of mediators from them, under the influence of which tissue infiltration occurs cellular elements and local edema. After 5 days in a sensitized animal, 40 μl of a suspension of the test solution of decamethoxin in the Hanks solution was injected into the small pad of the hind right paw (experimental). In the pad of the left paw (control) is the equivalent volume of Hanks solution. After 24 hours, the local incendiary reaction was determined, which was evaluated by the difference in the mass of the experimental (Rd) and control (Rk) paws. A quantitative assessment of the reaction was described by the reaction index (IR) (see table 1 1).

Table 1 1

The study of allergic properties of 0.2 mass% solution

decamethoxin in the GTS reaction, Me (Q25; Q75)

The data obtained indicate that the introduction of a solution of decamethoxin at a dose of 1, 3 mg / kg and 13 mg / kg does not sensitize the animal organism and does not cause the development of allergic inflammation, which proceeds according to a delayed type of hypersensitivity. Thus, in the GST reaction in mice, it was found that the solution of decamethoxin does not exhibit allergenic properties upon parenteral administration.

Therefore, decamethoxin in the form of 0.02 mass% and 0.2 mass% solutions for oral administration does not exert a local irritating effect on the mucous membranes of laboratory animals with local and oral administration in a therapeutic dose. Analysis of the results of studying the potential allergenic effect of decamethoxin in the form of 0.02 %% - 0.2 mass %% solutions for oral administration indicates the absence of allergenic properties.

4. Studies were carried out on the parameters of chronic and specific toxicity of decamethoxin for prolonged enteral administration, including the effect on the immune system, mutagenic, gonado, embryotoxic, and teratogenic properties.

The study of chronic toxicity when administered orally in doses of 3 ml / kg (0.6 mg / kg decamethoxin, conditionally therapeutic dose) and 10

eighteen

FIXED SHEET (RULE 91) ISAUA times it exceeds - 30 ml / kg (6 mg / kg decamethoxin) for 1 month was performed on outbred rats of both sexes (n = 36) with the study of external integral indicators, clinical and laboratory parameters (hematological, blood biochemistry, blood coagulation, renal excretory function, pathomorphological studies). The research results are presented in tables 12-28.

Table 12

Lethal results in laboratory rats after intragastric administration of 0.2 mass% decamethoxin solution

with prolonged observation

Table 13

The effect of 0.2 mass% decamethoxin solution on body weight (g) of male rats (X ± S _x )

Table 14

The effect of 0.2 mass% decamethoxin solution on body weight (g)

female rats (X ± S _x )

Note: * - deviations are significant relative to the output, p <0.05.

Table 15

The effect of 0.2 mass% decamethoxin solution on the functional state of NS of male rats in open field test (X (X _mn ^ X _{m! 1X} )

Table 16

The effect of 0.2 mass% decamethoxin solution on the functional state of the central nervous system of female rats in

test field open {X {X _rm + _max )

Table 17

The effect of 0.2 mass% decamethoxin solution on hematological parameters in male rats (X (X _{tm max} ), X ± S _x )

Table 18

The effect of 0.2 mass% decamethoxin solution on hematological parameters in female rats (X (X _rm - _max ), χ ± s _x )

Table 19

The effect of 0.2 mass %% decamethoxin solution on the biochemical parameters of blood serum in male rats (X ± S _x )

Note. ^* - Deviations are significant relative to control, p <0.025. Table 20

The effect of 0.2 mass% decamethoxin solution on the biochemical parameters of blood serum in female rats (X ± S _x )

Table 21

The effects of 0.2 mass% decamethoxin solution

on hemostasis in male rats (X ± S _x )

Table 22

The effects of 0.2 mass% decamethoxin solution

on hemostasis in female rats (X ± S _x )

Table 23

The influence of 0.2 mass% decamethoxin solution on the functional state of the kidneys in male rats in urine (X ± S _x )

Table 24

The effect of 0.2 mass% decamethoxin solution on the functional state of the kidneys in female rats in urine (X ± S _x )

Table 25

The effect of 0.2 mass% decamethoxin solution on ECG in male rats (X ± S _x )

Table 26

The effect of 0.2 mass% decamethoxin solution on ECG in female rats (X ± S _x )

Table 27

The influence of 0.2 mass% decamethoxin solution on the ratios of the masses of internal organs in male rats (X (X _min + X _max )

Table 28

The influence of 0.2 mass% decamethoxinan solution ratios of the mass of internal organs in female rats (X ( _min - X _so )

Output. The results of the study showed that the oral administration of a conventional therapeutic dose of 3 ml / kg (0.6 mg / kg for decamethoxin) and a dose that is 10 times higher than 30 ml / kg (6 mg / kg for decamethoxin) does not cause morphological signs cardiotoxic, nephrotoxic, hepatotoxic effects on the body of experimental animals, does not cause reactive changes in the adrenal cortex, does not violate the state of the lung tissue, does not affect the morphofunctional state of the immune system, reproduction, sensorimotor cortex b cerebral hemispheres and does not adversely affect the internal organs of animals with other studied indicators.

Studies of the cumulative effect of oral administration were performed on outbred rats of both sexes (n = 12), to whom the drug was administered for 28 days in increasing doses from the initial dose (0.1 LD50), which was 60 mg / kg. Every four days the dose of the drug was increased by 0.1; 0.15; 0.22; 0.34; 0.5; 0.75; 1, 12 times from LDW (600 mg / kg for rats). The results are shown in tables 29 and 30.

Output. The results of studying the cumulative effect of 0.02 mass% and 0.2 mass% solutions of decamethoxin indicate the absence of animal death, which indicates not only the absence of cumulative properties of the drug, but also the development of adaptive processes that result from prolonged administration of the studied pharmaceutically active substance contribute to the survival of animals with a dose that exceeds LD50.

27

FIXED SHEET (RULE 91) ISAUA Table 29

Determination of the cumulative effect of 0.2 mass% decamethoxin solution following the Lim method. The death of animals depending on

total dose

Table 30

The dynamics of body weight of animals in the study of the cumulative effect

0.2 mass% decamethoxin solution (X ± S _x )

Note: * - the deviation is reliable relative to the initial indicators; 0.05.

Studies of the mutagenic effect were carried out on outbred mice of both sexes (n = 24) with a single oral dose of 5 ml / kg (1 mg / kg) and 50 ml / kg (10 mg / kg) for 6, 24 and 48 hours from experience with further counting of bone marrow cells from chromosome aberrations in the metaphase stage. 2 hours before the animals were removed from the experiment, a 0.025% colchicine solution was intraperitoneally injected in order to accumulate cells in the separation metaphase. Both femurs were taken from each animal, from which the bone marrow was washed. The results are shown in table 31.

Output. Five-day intragastric administration of 0.02 mass %% and 0.2 mass %% solutions of decamethoxin in doses of 5 ml / kg and 50 ml / kg to male mice stimulated an increase in the number of aberrant cells, and also did not cause aberrations of chromosomal and chromatid types. Thus, the results of a quantitative assessment of the cytogenetic activity of decamethoxin in bone marrow cells of male mice with an intragastric route of administration indicate the absence of mutagenic properties.

The study of the immunotoxic effect of the drug was carried out when administered orally on outbred mice (n = 70) by evaluating the effect on humoral and cellular immunity.

The study of the toxic effect on cellular immunity was carried out on 40 mature mice (n = 40) in the test “Slow Type Hypersensitivity Reaction” (GTS) using the Kitamura Raman method. The reaction is aimed at determining the ability of the test substance to influence the production of sensitized T-lymphocytes-mediator effectors, which

29th

FIXED SHEET (RULE 91) ISAUA cause tissue infiltration by cellular elements.

Table 31

The results of the assessment of cytogenetic activity _. 0.2 mass% decamethoxin solution in tests taking into account chromosomal aberrations in cells

bone marrow of male mice, Me (Q25; Q75) (n = 6)

Notes:

1. p1 - level of significance of differences with respect to intact control, χ ²;

2. p2 - level of significance of differences with respect to the negative control, χ ²;

3. p - the number of observations.

The introduction of antigen into the paw pad of an animal leads to the development of local edema. 0.02 mass% and 0.2 mass% decamethoxin solutions were administered intragastrically once a day every hour after and during the entire immunization period (5 days) to the day the results were taken into account at a conventional therapeutic dose of 6.5 ml / kg (1.3 mg / kg for decamethoxin) and 10 times more - 65 Ml / kg (13 mg / kg for decamethoxin).

Animals were immunized with a single administration. suspensions

thirty

FIXED SHEET (RULE 91) ISAUA freshly washed sheep red blood cells (EB). On the 5th day, in order to detect immunization in mice, a final dose of antigen was administered under the aponeurotic plate of one of the hind limbs. Hanks solution was injected into the contralateral paw. After 24 hours, the cut off paws were weighed in animals removed from the experiment, on the basis of which a conclusion was drawn on the degree of limb edema as a result of a delayed-type hypersensitivity reaction. The results of a study of the effect of decamethoxin on the functional activity of T-cells-effectors in a delayed-type hypersensitivity reaction are shown in table 32.

Table 32

The effect of 0.2 mass% decamethoxin solution on the primary cellular immune response of mice immunized with sheep erythrocytes

The nature of the influence of 0.02 mass% and 0.2 mass% decamethoxin solutions on the humoral immune response was evaluated by the number of antibody-forming cells (AOK) in the spleen and hemagglutinin (A) titers in the blood serum of 30 immunized mice. On the 5th day after immunization, the amount of AOK in the spleen was determined, in blood serum - titers A. The amount of AOK in the spleen was determined using the method of local hemolysis in gael. This method is based on the ability of lymphoid cells of experimental animals immunized with foreign red blood cells to secrete anti-erythrocyte antibodies that cause red blood cell lysis in the presence of complement.

The number of producers of antibodies to the lymphoid organ was determined by the number of macroscopically visible hemolysis zones around the antibodies of the forming cells. Titers A in the blood serum of immunized animals were determined by serial dilution in polystyrene plates. The agglutination reaction is based on the ability of antibodies (agglutinins), which are contained in the blood serum of immunized animals, to adhere ram sheep red blood cells used as antigens. The results of determining the potential immunotoxic properties of decamethoxin are shown in table 33.

31

FIXED SHEET (RULE 91) ISAUA Thus, on the basis of the conducted studies, it can be concluded that decamethoxin in the form of 0.02 mass% and 0.2 mass% solutions upon intragastric administration to mice at a conditionally therapeutic dose of 6.5 ml / kg (1, 3 mg / kg for decamethoxin) and 10 times its excess - 65 ml / kg (13 mg / kg for decamethoxin) - does not have an immunotoxic effect on the humoral and cellular immune response of mice.

Table 33 The effect of 0.2 mass% decamethoxin solution on the primary humoral immune response of mice immunized with sheep erythrocytes

The study of gonadotoxicity of 0.02-0.2 mass% solution of decamethoxin was carried out in 72 rats (40 males and 32 females) by oral administration of the drug once a day at doses of 3 and 30 ml / kg for 15 days, which corresponds to 3 astral cycles females, with further study of the functional state and microstructure of the ovaries of females and testes of male rats. The results of the study are presented in tables 34-36.

Table 34

Duration of the astral cycle in female rats injected with 0.2

mass% solution of decamethoxin in doses of 3 and 30 ml / kg,

Me (Q25; Q75) (n = 8)

Table 35

Macrometric indicators of the ovaries of female rats that were injected with 0.2 mass% decamethoxin solution at doses of 3 and 30 ml / kg, M ± m (n = 5)

Table 36

The composition of the structural and functional elements of the ovaries of female rats that received 0.2 mass% decamethoxin solution (n = 5)

Notes: 1. * - statistically significant differences relative to intact control, ^* * - negative control, Mann-Whitney test (p ≤ 0.05), 2. p - number of animals in each group

Output. It was found that intragastric administration of 0.02 mass %% and 0.2 mass %% solutions of decamethoxin to females at a therapeutic dose of 3 mg / kg does not negatively affect the structural organization of the ovaries, does not reduce the reserve of folliculogenesis, does not affect the rate of development of egg follicles, does not increase follicular atresia. But at a dose of 30 ml / kg, which is 10 times higher than the conditionally therapeutic, decamethoxin helps to reduce the number of primordial and growing follicles, and leads to a slight decrease in the mass coefficients of the ovaries, which can be regarded as a manifestation of the gonadotoxic effect in females.

The results of a study of gonadotoxicity in males are presented in tables 37-39.

Table 37

Macrometric indicators of testes of rats, which were intragastrically injected with 0.2 mass% solution of decamethoxin in doses of 3 and 30 ml / kg,

M ± m (n = 10)

Output. Analyzing the data obtained, it can be stated that a 0.2 mass %% solution of decamethoxin in both doses studied does not negatively affect both the functional state of spermatozoa, the structure of testicular tissue and the process of spermatogenesis, that is, it does not have a gonadotoxic effect on male gonads.

34

FIXED SHEET (RULE 91) ISAUA Table 38

Indicators of the functional state of rat sperm, which was administered intragastric 0.2 mass% decamethoxin solution in doses of 3 and 30 ml / kg (n = 10)

Table 39

Morphometric indicators of the process of spermatogenesis in rats to which intragastric was administered 0.2 mass% of a solution of decamethoxin in doses of 3 and 30 ml / kg

The reproductive toxicity of the substance was studied in 60 male rats and 120 females with the following determination of the effect of decamethoxin at the progenesis stage: the formation of male and female gametes, violation of sexual behavior and transport of conception products. The results of studies on males are presented in tables 40-49

Table 40

Fertilization rates in males that received 0.2 weight% decamethoxin solution when mating with intact females

Table 41

Indicators that are recorded at autopsy of pregnant females on the 20th day of gestation, fertilized by males who were injected with 0.2 mass% decamethoxin solution at doses of 3 and 30 ml / kg

36

FIXED SHEET (RULE 91) ISAUA Table 42

The indicators of embryo action with the introduction of 0.2 mass% solution of decamethoxin in doses of 3 and 30 ml / kg

Table 43

The size of offspring and the gender of the offspring of females fertilized by males who were injected with 0.2 mass% solution of decamethoxin in doses of 3 and 30 ml / kg, Me (LQ; UQ)

37

FIXED SHEET (RULE 91) ISAUA Table 44

The body mass dynamics of the offspring of females fertilized by males who were injected with 0.2 mass% decamethoxin solution in doses

3 and 30 ml / kg, M ± t

Note: ^* - statistically significant changes relative to the output, ≤ 005

The results of the study on females are presented in tables 45-49.

Table 45

Fertilization rates in females that received 0.2 weight% decamethoxin solution when mating

with intact males

Table 46

Indicators that are recorded at autopsy of pregnant females on the 20th day of gestation, which were injected with 0.2 mass% solution of decamethoxin in doses of 3 and 30 ml / kg

Indicators of embryo action of females who were injected

0.2 mass% decamethoxin solution in doses of 3 and 30 ml / kg

39

FIXED SHEET (RULE 91) ISAUA Table 48

The size of the offspring and the gender of the offspring of the females, which were introduced 0.2 mass% solution of decamethoxin in doses of 3 and 30 ml / kg,

Me (LQ; UQ)

Table 49

The body mass dynamics of the offspring of females fertilized by males who were injected with 0.2 mass% solution of decamethoxin in doses of 3 and 30 ml / kg, M ± t

Note: * - changes are statistically significant relative to the output, ≤ 0.05

Output. A study of the effect of oral administration of 0.02 mass% and 0.2 mass% of decamethoxin solutions in doses of 3 and 30 ml / kg for 60 days in males and 15 days in female rats showed no negative effect on the reproductive function of animals.

Studies of embryotoxicity and teratogenicity were carried out at 160 outbred rats (40 males and 120 females) by oral administration of 0.02 mass% x decamethoxin solution at a conventional therapeutic dose of 3 ml / kg and at a dose that corresponds to 10 times (maximum dose) - 30 ml / kg,. with the following definition of embryotoxicity parameters (embryoletal effect, developmental disability: changes in body weight, cranio-caudal size of the fetus, delayed skeletal ossification) and teratogenicity (presence of visible external developmental anomalies and hematomas, anomalies of the skeletal system and ossification centers). The results are presented in tables 50-53.

The results of the study showed the absence of embryotoxic action of 0.02 mass% and 0.2 mass% of decamethoxin solutions in a therapeutic dose of 3 ml / kg. The introduction of a toxic dose of 0.02 mass% and 0.2 mass% decamethoxin solutions of 30 ml / kg, 10 times higher than the therapeutic, had a negative effect on embryos in the first trimester of gestation. This was manifested in an increase in preimplantation mortality, and when introduced during implantation and organogenesis (6-16 days), which was expressed in a decrease in the weight of the fetuses and their cranio-caudal size. The teratogenic effect of decamethoxin was established when it was administered in both doses during implantation and organogenesis, which suggests that the drug should not be used to treat intestinal infections in pregnant women.

Table 50

Indicators that are recorded at the opening of pregnant females on the 20th day of gestation, after the introduction of 0.2 mass% solution of decamethoxin in doses of 3 and 30 ml / kg at different periods of pregnancy

41

FIXED SHEET (RULE 91) ISAUA amount

9.0 9.5 live 9.0 10.0 9.0 9.0

(7.0; (9.0; fruits, Me (8.0; 1 1, 0) (9.0; 11, 0) (8.0; 10.0) (8.0; 10.0)

10.0) 1.0) (LQ; UQ)

amount

dead 0 0 0 0 0 0 fruits, Me (0; 0) (0; 0) (0; 0) (0; 0) (0; 0) (0; 0)

(LQ; UQ)

Craniocaudal

31, 4 ± 0.4 31, 7 ± 0.4 29.6 ± 0.8 30.0 ± 0.4 29.2 ± 0.4 * ^* 29.7 ± 0.6 size, mm,

M ± t

Weight

fruit, g, 2.59 ± 0.05 2.55 ± 0.05 2.51 ± 0.13 2.32 ± 0.06 2, 19 ± 0.06 * / * ^* 2.29 ± 0.08

M ± t

Weight

placenta, g, 0.62 ± 0.01 0.65 ± 0.03 0.67 ± 0.03 0.60 ± 0.01 0.61 ± 0.04 0.57 ± 0.01

M ± t

Fetal placental 0.24 ± 0.00 0.25 ± 0.01 0.27 ± 0.01 0.26 ± 0.01 0.28 ± 0.02 0.25 ± 0.01 index, M ± t

Notes: 1. ^* - statistically significant differences when compared with the intact control group, Newman-Keyles test (p ≤ 0.05),

2. ^** - statistically significant differences when compared with the negative control group, Newman-Cales test (p ≤ 0.05)

Table 51

Indicators of embryo action with the introduction of 0.2 mass% decamethoxin solution at doses of 3 and 30 ml / kg at different periods of pregnancy

Note: ^* - statistically significant differences when compared with the intact control group, Mann-Whitney test (p ≤ 0.05).

42

FIXED SHEET (RULE 91) ISAUA Table 52

The state of the internal organs of the fetuses of females on the 20th day of gestation, after intragastric administration of 0.2 mass% of a solution of decamethoxin in doses of 3 and 30 ml / kg at different periods of pregnancy,

Me (LQ; UQ)

The state of the skeletal system of the fetuses of females on the 20th day of gestation,

after intragastric administration of 0.2 mass% solution

decamethoxin in doses of 3 and 30 ml / kg during different periods of pregnancy

43

FIXED SHEET (RULE 91) ISAUA sublingual

bones, %,

M (Q25; Q75)

Delay

ossification in 0 0 0 0 0 0 pubic bone, (0; 0) (0; 0) (0; 6) (0; 0) (0; 0) (0; 0)%, M (Q25; Q75)

Lack of

ossification in 0 0 10 45 40 20 of the pubic bone, (0; 0) (0; 20) (0; 33.3) (10; 77.5) (0; 80) (0; 33.3)

%, M (Q25; Q75)

Delay

ossification

0 0 0 0 0 0 sciatic

(0; 0) (0; 0) (0; 0) (0; 10) (0; 0) (0; 0) bones,%,

M (Q25; Q75)

Lack of

ossification

0 0 0 0 0 0 sciatic

(0; 0) (0; 0) (0; 0) (0; 10) (0; 50) ^** (0; 20) bones,%,

M (Q25; Q75)

Delay

ossification

0 0 0 0 0 0 occipital

(0; 0) (0; 0) (0; 0) (0; 0) (0; 0) (0; 0) bones,%,

M (Q25; Q75)

Lack of

ossification

0 0 0 0 0 0 occipital

(0; 0) (0; 0) (0; 0) (0; 0) (0; 50) (0; 33) bones,%,

M (Q25; Q75)

Delay

ossification

0 0 0 0 0 0 cross-dark

(0; 0) (0; 0) (0; 0) (0; 12) (0; 40) (0; 16) bones,%,

M (Q25; Q75)

Lack of

ossification

0 0 0 0 0 0 cross-dark

(0; 0) (0; 0) (0; 0) (0; 0) (0; 33) (0; 20) bones,%,

M (Q25; Q75)

Delay

ossification 0 0 0 10 0 0 parietal bone, (0; 20) (0; 0) (0; 40) (0; 37) (0; 75) (0; 33)

%, M (Q25; Q75)

Lack of

ossification 0 0 0 0 0 0 parietal bone, (0; 0) (0; 0) (0; 0) (0; 0) (0; 0) (0; 0)%, M (Q25; Q75)

Delay

ossification 0 0 0 0 0 0 frontal bone, (0; 0) (0; 0) (0; 0) (0; S) (0; 50) (0; 0)

%, M (Q25; Q75) Lack of

ossification in

arcs

vertebrae 0 0 0 0 0 0 lumbar (0; 0) (0; 0) (0; 0) (0; 12) (0; 20) (0; 0) department

the spine

%, M (Q25; Q75)

Lack of

ossification in

bodies

vertebrae 0 0 0 0 0 0 lumbar (0; 0) (0; 0) (0; 0) (0; 0) (0; 20) (0; 0) department

the spine

%, M (Q25; Q75)

Lack of

ossification in

arches of the vertebrae 50 75

17 0 29 33 sacral (0; 100) (40; 100)

(0; 20) (0; 40) (0; 60)

department _* ι _** (0; 83) of the spine,

%, M (Q25; Q75)

Lack of

ossification in

vertebral bodies 66

0 0 0 29 20 sacral (20; 100)

(0; 0) (0; 28) (0; 20) (0; 60) ^* (0; 80) department

the spine

%, M (Q25; Q75)

amount

centers

ossification in

lumbar 17.9 ± 0.02 17.9 ± 0.05 17.5 ± 0.46 17.2 ± 0.47 17.2 ± 0.39 17.4 ± 0.35

the spine

M ± t

amount

centers

ossification in

8.0 ± 0.83 7.74 ± 0.80 sacral 10.97 ± 0.50 9.89 ± 0.75 9.81 ± 0.78 * * 9.26 + 0.78 division

the spine

M ± t

amount

centers 1, 64 ± 0.37 1, 57 ± 0.27

3.35 ± 0.28 3.20 + 0.30 2.85 ± 0.32 * * 2.41 ± 0.36 ossification in

breast, M ± t

The presence of the 14th

rudimentary 0 0 0 0 0 0 edges,%, (0; 0) (0; 0) (0; 0) (0; 0) (0; 0) (0; 20)

M (Q25; Q75)

Shortening 13- 0 0 0 0 0 0 ribs,%, (0; 0) (0; 0) (0; 0) (0; 0) (0; 0) (0; 0)

M (Q25; Q75)

Magnification 60

0 0 0 12 0 crown,%, (0; 80)

(0; 20) (0; 16) (0; 20) (0; 70) (0; 33) M (Q25; Q75)

Arc merging, 0 0 0 0 0 0

%, M (Q25; Q75) (0; 0) (0; 20) (0; 0) (0; 0) (0; 0) (0; 0)

Lack of

0 0 0 0 0 0 chest arches,%,

(0; 0) (0; 0) (0; 0) (0; 0) (0; 0) (0; 0) M (Q25; Q75)

Break in

0 0 0 0 0 0 tape,%,

(0; 0) (0; 0) (0; 0) (0; 0) (0; 0) (0; 0) M (Q25; Q75)

Note: ^* - statistically significant differences with intact,

- with negative control, Mann-Whitney test (p ≤ 0.05). Microorganisms such as Pseudomonas aeruginosa, Burgholderia cepacia, Staphylococcus aureus, E. coli, C. albicans, Klebsiella pneumoniae cause the following gastrointestinal diseases:

Pseudomonas aeruginosa enteritis, colitis, dysbiosis, enterocolitis;

- Burgholderia cepacia: peritonitis;

Staphylococcus aureus: diarrhea, coloproctitis;

E.coli: dysbiosis, escherichiosis;

C. albicans: candidiasis;

Klebsiella pneumoniae: dysbiosis, gastritis, gastroduodenitis, acute enteritis, enterocolitis.

To determine the effectiveness of using decamethoxin orally for the treatment of gastrointestinal diseases caused by marked microorganisms, the authors conducted the following studies:

1. Studies were carried out on the specific antimicrobial activity of decamethoxin at several research bases: a) In vitro studies of different concentrations of decamethoxin were performed on 8 museum strains of microorganisms and 14 strains of clinical isolates by serial dilutions in solid nutrient medium and by diffusion in agar.

The results of the study are presented in tables 54-62.

46

FIXED SHEET (RULE 91) ISAUA Table 54

The effect of different concentrations of decamethoxin (in mass%) on Pseudomonas aeruginosa

“+” - intensive growth of microorganisms;

"+" - slight growth (single colonies);

“-” - lack of growth;

Table 55 The effect of different concentrations of decamethoxin (in mass%)

at burgholderia cepacia

Table 56

The effect of different concentrations of decamethoxin solution (in mass%) on S. aureus, E. coli, C. albicans

Table 57

Establishment of the antimicrobial activity of different concentrations of decamethoxin solution (in mass%) in relation to clinically isolated strains of Pseudomonas aeruginosa by agar diffusion ("hole method")

Nsn / n Type of microorganism Growth retardation zone (in mm) for different

decamethoxin concentrations

0.02% 0.05% 0.1% 0.2% Control

1 2 3 4

1 P. aeruginosa 2293 0 14 16 18 0

2 P. aeruginosa 1299 14 16 18 20 0

3 P. aeruginosa 1327 12 15 18 19 0

4 P. aeruginosa 1642 15 16 18 20 0

5 P. aeruginosa 1645 13 14 16 17 0

6 P. aeruginosa 1644 12 15 19 21 0

7 P. aeruginosa 2268 13 16 20 23 0

8 P. aeruginosa 2306 13 16 19 21 0

9 P.aeruginosa 6756 0 13 16 18 0

10 P. aeruginosa ATCC 27853 15 17 19 20 0

11 P. aeruginosa 6,218 15 17 20 22 0 Table 58

The establishment of antimicrobial activity of different concentrations of decamethoxin solution (in mass%) in relation to museum cultures

Burgholderia cepacia agar diffusion method ("hole method")

Table 59

Establishment of antimicrobial activity of different concentrations of decamethoxin solution (in mass%) in relation to clinically isolated strains of Staphylococcus aureus and museum cultures of S. aureus, E. coli, Candida albicans by agar diffusion ("hole method")

Table 60

Establishment of the “death time” of museum and clinically isolated strains of Pseudomonas aeruginosa depending on the concentration of decamethoxin (in mass%) but the time of its effect on the microorganism

Table 61

Establishment of the "time of death" of museum cultures of Burgholderia cepacia depending on the concentration of decamethoxin (in mass%) and

the time of its effect on the microorganism

Table 62

Establishment of the “time of death” of clinically isolated strains of Staphylococcus aureus, museum cultures of S.aureus, E.coli, Canda albicans depending on the time of influence

0.2 mass% decamethoxin solution per microorganism

Conclusions from the research.

1. Staphylococci and fungi of the genus Candida show the greatest sensitivity to a solution of decakmethoxin, and less - burkholderia. The most resistant were museum and clinical strains of pseudomonads.

2. Decamethoxin at a concentration of 0.02% of Maos active relative to staphylococci and fungi of the genus Candida; at a concentration of 0.05 mass% - relative to burkholderry and some strains of pseudomonas; at a concentration of 0.1 mass% - relative to all used microorganisms.

3. A correlation was established between the antimicrobial action of decamethoxin on different types of microorganisms and the time of influence of its solution. Upon contact with microorganisms for 24 hours, decamethoxin exhibits a pronounced bactericidal effect even on multiresistant clinically isolated P.aeruganosa strains in a minimum concentration of 0.02 mass%.

b) A specific in vitro activity was studied by determining the minimum inhibitory concentration (MIC) of decamethoxin by serial dilution and sensitivity to decamethoxin by agar diffusion on 6 strains of microorganisms and in vivo studies (on 64 rats) on 2 models of infectious coloproctitis caused by Pseudomonas aeruginosa and Staphylococcus aureus with the following definition of external, clinical, laboratory, microbiological parameters.

The results of determining the minimum inhibitory concentration (MIC) of the minimum bactericidal concentration (MBC) of 0.02 %% solution

51

FIXED SHEET (RULE 91) ISAUA decamethoxin by serial dilution is presented in table 63.

Table 63

Characterization of antimicrobial activity of 0.02 mass% decamethoxin solution by multiple serial dilutions

The results of the study of the antimicrobial activity of decamethoxin by the well method are shown in table 64.

Table 64

Characterization of the antimicrobial activity of 0.02 mass% decamethoxin solution due to the effect on the growth zone of microorganisms by the method

diffusion in agar (wells)

According to the results of the studies, it was found that decamethoxin in the form of a solution showed pronounced antimicrobial activity, relative to gram-positive (S. aureus, S. pyogenes, B.subtilis), and gram-negative (E.coli, K. pneumoniae) microorganisms. In relation to P. aeruginosa, antimicrobial activity is less pronounced. Relative to fungi of the genus Candida, decamethoxin showed pronounced fungistatic activity. Thus, decamethoxin exhibits pronounced antimicrobial activity in vitro and may be recommended for the treatment of intestinal infections.

2. Studies have been conducted on the effectiveness of decamethoxin in vivo in models of infectious coloproctitis in rats (n = 32) caused by S. aureus and P. aeruginosa, with morphological verification compared to Nifuroxazide.

The results of studies on a model of coloproctitis caused by S. Aureus, are presented in table 65-70.

Table 65

Antimicrobial activity study design of a 0.02 mass% decamethoxin solution in a S. aureus model of infectious coloproctitis in rats

Table 66

Dynamics of rats body mass under conditions of S. aureus infectious coloproctitis under the influence of decamethoxin and nifuroxazide

Notes.

PKI - intact (operated) control, KP - control pathology.

* - Deviations are significant relative to the PKI, p 0, 05.

* ^* - Deviations are significant relative to KP, p <0.05

53

FIXED SHEET (RULE 91) ISAUA Table 67

The dynamics of the degree of infection of rat feces under conditions of infectious coloproctitis caused by S. aureus under the influence of 0.02 mass% solution of decamethoxin and Nifuroxazide

Note. PKI - intact (operated) control, KP - control pathology.

Table 68

Hematological parameters of rats under conditions of infectious coloproctitis caused by S. aureus under the influence of 0.02 mass% solution of decamethoxin and Nifuroxazide (X (X _min - - X _so ), X ± S _x )

PKI - intact (operated) control, KP - control pathology.

* - Deviations are significant relative to the operated control, p <0.05

** - Deviations are significant relative to the control pathology, p <0.05.

*** - Deviation significant relative to the drug comparison, p <0.05.

54

FIXED SHEET (RULE 91) ISAUA Table 69

The rat hemostasis in infectious coloproctitis caused by S. aureus under the influence of 0.02 mass% solution

decamethoxin and nifuroxazide

Note. PKI - intact (operated) control, KP - control pathology

Table 70

Biochemical parameters of rat blood serum in infectious coloproctitis caused by S. aureus under the influence of 0.02 mass% solution of decamethoxin and nifuroxazide (X ± S _x )

Notes.

PKI - intact (operated) control, KP - control pathology.

* - Deviations are significant relative to the PKI, p <0.05.

** - Deviations are significant relative to KP, p <0.05.

# - Deviation significant relative to the drug comparison, p <0.05.

Under the conditions of infectious coloproctitis caused by S. aureus, when administered 0.02 mass%, a solution of decamethoxin exhibits pronounced antimicrobial properties that exceed the antimicrobial activity of Nifuroxazide and contribute to the restoration of the functional state of the animal organism.

So, unlike Nifuroxazide, decamethoxin causes

55

FIXED SHEET (RULE 91) ISAUA significant in comparison with the control pathology group, an increase of 1 1% of animal body weight.

The pronounced antimicrobial activity of decamethoxin is confirmed by a decrease, compared with the control pathology group, by almost an order of magnitude (7.5 times) in the number of colony forming units of the pathogen on the 6th day of the experiment, while under the influence of Nifuroxazide this indicator remains at the level of the control pathology group.

With the severity of the antimicrobial action, decamethoxin exceeds the reference nifuroxazide by 7.4 times. The therapeutic effect and antimicrobial effect of decamethoxin is evidenced by the reliable renewal of leukocyte count indicators: eosinophils, lymphocytes and monocytes, as compared with the control pathology group. Decamethoxin has a noticeable positive effect on the development of the pathological process in the large intestine of rats. It helps to clean the surface of the mucous membrane from foci of colonization of microorganisms, prevents the destruction of the surface epithelium, reduces incendiary cell infiltration of the own plate of the mucous membrane. The therapeutic effect of decamethoxin is superior to the comparison drug "Nifuroxazide" in the model of infectious coloproctitis caused by S. Aureus.

The results of studies on a model of infectious coloproctitis caused by P. aeruginosa are presented in tables 71 -76.

Table 71 Design study of the antimicrobial activity of 0.02 mass% solution of decamethoxin in a model of infectious coloproctitis in rats caused by P. aeruginosa

56

FIXED SHEET (RULE 91) ISAUA Table 72

The dynamics of rat body mass under conditions of infectious coloproctitis caused by P. aeruginosa under the influence of 0.02 mass% solution of decamethoxin and Nifuroxazide

Notes.

PKI - intact (operated) control, KP - control pathology.

* - Deviations are significant relative to the PKI, p <0.05.

* ^* - Deviations are significant relative to KP, p <0.05.

Table 73

The dynamics of the degree of infection of rats in infectious coloproctitis caused by P. aeruginosa, under the influence of 0.02 mass% solution

decamethoxin and nifuroxazide

Note. PKI - intact (operated) control, KP - control pathology Table 74

Hematological indices in rats koloproktita infectious conditions caused by P. aeruginosa, under the influence of 0.02% by weight solution and decamethoxin nifuroxazide (X _(tt - x _s), X ± S _x)

Notes.

PKI - intact (operated) control, KP - control pathology.

* - Deviations are significant relative to the operated control, p <0.05.

** - Deviation significant relative to pathology control, p <0.05.

*** - Deviation significant relative to the drug comparison, p <0.05.

Table 75

Rat hemostasis of rats under conditions of infectious coloproctitis caused by P. aeruginosa under the influence of 0.02 mass %% solution of decamethoxin and nifuroxazide

Note.

PKI - intact (operated) control, KP - control pathology.

* - Deviations are significant relative to the operated control, p <0.05. Table 76

Biochemical parameters of rat blood serum under conditions of infectious coloproctitis caused by P. aeruginosa under the influence of 0.02 mass% solution of decamethoxin and Nifuroxazide (X ± S _x )

Notes.

PKI - intact (operated) control, KP - control pathology.

* - Deviations are significant relative to the PKI, p <0.05.

** - Deviations are significant relative to KP, p <0.05.

# - Deviation significant relative to the drug comparison, p <0.05.

Similar results were obtained in the study of concentrations up to 0.2 mass% decamethoxin.

Under the conditions of infectious coloproctitis caused by P. aeruginosa, when administered orally, 0.02-0.2 mass% of a solution of decamethoxin exhibits pronounced antimicrobial properties that exceed the antimicrobial activity of Nifuroxazide and contribute to the restoration of the functional state of the animal organism. So, unlike Nifuroxazide, decamethoxin causes a significant increase of 1 1% of the animal’s body weight compared to the control pathology group. Hyperfibrinogenemia with both decamethoxin and Nifuroxazide did not lead to an increase in blood coagulation, since the coagulation time did not change. The pronounced antimicrobial activity of decamethoxin is confirmed by a 1.8-fold decrease in the number of colony forming units against the control pathology group on the 6th day of the experiment, while under the influence of Nifuroxazide this indicator remains at the level of the control pathology group. With the severity of the antimicrobial action, decamethoxin exceeds the reference Nifuroxazide by 54 times. About therapeutic effect decamethoxin indicates reliable, compared with the control pathology group, the renewal of leukocyte count indicators: stab neutrophils and eosinophils. The results of morphological studies indicate that decamethoxin has a pronounced therapeutic effect on the development of the pathological process in the large intestine of rats. It helps to clean the surface of the mucous membrane from the foci of colonization of microorganisms, reduces incendiary cell infiltration of its own plate of the mucous membrane, and prevents the destruction of the surface epithelium. Decamethoxin surpasses the reference drug "Nifuroxazide" in a beneficial effect on the developed experimental pathology - coloproctitis caused by Pseudomonas aeruginosa.

3. A study was conducted to determine the antimicrobial activity of decamethoxin in vitro on strains of microorganisms of the intestinal group (S. aureus, P. aeruginosa and E. coli., C. albicans, S. paratyphi) at different pH values, the effect of decamethoxin on film formation and biomass accumulation

The results of the study are presented in tables 77-79.

Table 77

Antibacterial activity of decamethoxin solution

Table 78

Antifungal activity of decamethoxin solution

Table 79

The diameters of the zones of growth inhibition of microorganisms in the presence of decamethoxin under the condition of different pH values (M ± W)

Note: "-" - there is no growth inhibition zone;

'# "- no studies have been conducted.

Findings. with the results of research.

1. It was found that the decamethoxin solution has inherent pronounced antimicrobial properties in relation to P. aeruginosa, E. coli, S. aureus, S. paratyphi and C. albicans. The severity of the inhibitory effect of a decamethoxin solution depends on the type of microorganism and pH. The antibacterial activity of decamethoxin relative to test strains of bacteria and fungi was enhanced in an alkaline environment. .

2. Decamethoxin in all studied in different doses (10.0 μg, 25.0 μg, 50.0 μg 100.0 μg) exhibits pronounced antimicrobial properties. The diameters of growth inhibition zones of test microorganisms vary depending on the type of microorganism and pH. The highest activity of decamethoxin solution in relation to P. aeruginosa, S. aureus, E. coli

61

FIXED SHEET (RULE 91) ISAUA observed at pH 8.0  0.1, relative to C. albicans - at 7.15  0.2, with respect to S. paratyphi at pH values of 8.0  0.1 and 5, 1  0.2.

3. A solution of decamethoxin dose-dependently violates the film formation of microorganisms. According to the degree of inhibitory effect with respect to the biofilms of P. aeruginosa and E. coli, the activity of the decamethoxin solution under study changed depending on the pH value. The most pronounced inhibitory effect of decamethoxin is recorded at a pH of 8.0  0.1 at a concentration of 10.0 MIC.

4. It was found that the formation of biomass of microorganisms under the action of decamethoxin depends on the concentration of the substance, exposure time and pH of the medium. Inhibition of the growth of E. coli biomass is observed after 1 hour at pH values of 8.0  0.1 and increases with increasing incubation time. Pronounced inhibitory effect against S. paratyphi _: observed after 24 and 48 hours in an acidic and alkaline environment. Inhibition of growth and reproduction of P. aeruginosa is observed starting from 6 hours of decamethoxin action at a pH of 6.3 0,4 0.4 and increases with increasing incubation time. S. aureus exhibits a sensitivity to decamethoxin in the first hours of action (after 6 hours) at a pH of 5.1 17 0.216. After 24 and 48 hours, the most pronounced inhibition of the growth of Staphylococcus aureus biomass is recorded at a pH of 5.1 & 0.216 and a pH of 6.3 # 0.416. It was found that the pH value practically does not affect the sensitivity of C. albicans fungi to decamethoxin.

4. The main pathogenic human viruses can be divided into 3 categories according to the gastrointestinal tract, in which they are involved in pathological processes:

viruses that cause acute gastroenteritis, mainly in children: rotaviruses, caliciviruses, intestinal adenoviruses, astroviruses, toroviruses;

viruses that affect the mucous membrane of the colon: herpes simplex virus type 2 and cytomegalovirus;

viruses that cause changes in the epithelium of the anal canal and perianal skin, including the human papilloma virus.

Studies have been conducted on the cytotoxicity and virucidal effect of decamethoxin on certain types of viruses.

Cytotoxicity studies in cell cultures were carried out at two research bases:

1) Studied on a culture of human adenocarcinoma (HEP-2) and dog kidney cell line (MDCK); 2) Cytotoxicity was studied in 4 cultures of human cells: CFTE 29 (tracheal epithelium), AGS (epithelial cells of the stomach, adenocarcinoma), HGEPp.05 (epithelium is clear), HBER.05 (bladder epithelium) using the MTT test, NR- test, Bradford test.

The results of the study are presented in tables 80-84. Cd50

62

FIXED SHEET (RULE 91) ISAUA (cytotoxic concentration) was determined as such the concentration of the active substance decamethoxin in μg / ml, which causes a cytotoxic effect in half of the treated cell monolayers after 72 hours of observation. The minimum inhibitory concentration (MIC) is the lowest concentration of antibiotic (in mg / l or μg / ml), which in vitro completely inhibits the visible growth of microorganisms.

The results of a study of cytotoxicity of 0.02 mass% and 0.05 mass% of decamethoxin in a human adenocarcinoma (HEP-2) and dog kidney cell line (MDCK) culture showed that the sensitivity of cells to decamethoxin depends on the type of cell culture, and in terms of CDso and MPC in decamethocosin MDCK cell culture was several times less toxic than in HEP-2 culture.

The CDso decamethoxin in the NEP-2 culture was 3.213 μg / ml, and in the MDCK culture it was 12.5 μg / ml (the final concentration of decamethoxin taking into account dilution in the culture medium was 0.01 mass%, i.e., 2 times less than the minimum antiseptic concentration). The MPC of decamethoxin in the NEP-2 culture was 1.563 μg / ml, and in the MDCK culture it was 6.25 μg / ml. In both cell cultures, the cytotoxic effect of decamethoxin was fully realized after 24 hours of exposure and remained unchanged during 48 and 72 hours of observation.

The results of the study indicate that decamethoxin at a concentration of 0.05 mass% has a pronounced cytotoxic effect on the viability of the CFTE29o cell line starting from the exposure time of 30 seconds, decamethoxin at a concentration of 0.02 mass% - starting from the exposure time of 1 minute (P <0, 05 relative to the reference drug), decamethoxin at a concentration of 0.05 mass% - starting from the exposure time of 5 minutes (P <0.05 relative to the reference drug). Referent preparations Desin (0.02% and 0.05%), Miramistin (0.01%) and Xylometazoline (0.10%) also exhibit similar cytotoxic effects - an increase in the incubation period with these drugs up to 30 minutes leads to complete cell death CFTE29o lines. The drug Salbutamol (0.10%) showed the least cytotoxic effect. Decamethoxin at concentrations of 0.05 mass% and 0.02 mass% has significantly higher cytotoxicity relative to the corresponding concentration of chlorhexidine with a short-term effect (0.5 and 1 minutes on AGS cells. A study of the effect of decamethoxin on the viability of human epithelial cells of the HGEp and HBER lines showed that in concentrations of 0.02 mass% and 0.05 mass%, there was no significant difference between the cytotoxic effect of decamethoxin and Desin on the cells of the marked lines.

63

FIXED SHEET (RULE 91) ISAUA Table 80

The study of the cytotoxic effect of decamethoxin (0.02 mass% solution) in HEP -2 and MDCK cell cultures

64

FIXED SHEET (RULE 91) ISAUA Table 81

Realization of the cytotoxic effect of decamethoxin (0.02 mass% solution) depending on the duration of its effect on cell monolayers

Table 82

The study of the cytotoxic effect of decamethoxin

(0.05 mass% solution) in HEP - 2 cell cultures

Table 83

The study of the cytotoxic effect of decamethoxin

(0.05 mass% solution) in MDCK cell cultures

Table 84

Realization of the cytotoxic effect of decamethoxin (0.05 mass% solution) depending on the duration of its effect on cell monolayers

Studies of the virucidal effect of decamethoxin were also carried out on the cytomegalovirus and intestinal adenovirus. It was found that decamethoxin has a virucidal effect relative to the aforementioned viruses, completely inactivates their 100-1000 infectious doses with a duration of exposure of 5 minutes.

65

FIXED SHEET (RULE 91) ISAUA the virucidal effect of decamethoxin in relation to cytomegalovirus and intestinal adenovirus

To study the effect of decamethoxin when taken orally on the development of infectious diseases of the gastrointestinal tract, a study was conducted on patients.

The experiments performed showed an unexpected result, which consists in the positive results of the treatment of infectious coloproctitis with decamethoxin as a pharmaceutically active substance in vivo when administered orally.

We studied 2 groups of patients treating infectious coloproctitis:

Group 1 - treatment with surgical intervention

66

FIXED SHEET (RULE 91) ISAUA Group 2 - treatment without surgery ..

The study of the first group of patients treated with surgery:

The study involved 30 patients. All patients were diagnosed with acute infectious coloproctitis caused by the bacteria Staphylococcus aureus. Surgical treatment was prescribed for patients: during the operation, a purulent cavity, an abscess were opened, pus was evacuated and drainage was placed. Subsequently, the wound healed itself or stitches were placed on it depending on the size of the abscess.

To prevent the transition of the acute form of infectious coloproctitis to the chronic form, postoperative therapy was carried out: in the first days after the operation, patients were not fed, only liquid was given, starting from day 3, a diet was prescribed.

All patients included in the study were divided into 2 clinical groups (see tables 86-87) for 15 people: patients of groups 1 were prescribed Nifuroxazide as postoperative therapy, patients of group 2 were prescribed 0.02-0.2 mass% solution of decamethoxin orally. The research results are shown in tables 86-88.

Table 86

Gender distribution of patients

The average age of patients in the studied groups

Table 88

Dynamics of body weight of patients in conditions of infectious coloproctitis, kg

67

FIXED SHEET (RULE 91) ISAUA Based on the data in table 88, it is seen that, compared with the initial state, the average body weight of patients during treatment with Nifuroxazide did not change, and when treated with decamethoxin solution, the average body weight of the patient increased by 4%.

From the information noted above, it is clear that decamethoxin affects the course of the disease better than Nifuroxazide: wounds in the intestine heal better, assimilation of nutrients is established, which leads to a set of patients.

Table 89

The dynamics of the degree of infection of feces of patients in conditions of infectious coloproctitis caused by Staphylococcus aureus

Based on the data in table 89, it can be seen that the decamethoxin solution is better than the comparison tool Nifuroxazide affects the development of Staphylococcus aureus in the human intestine, reducing the number of colony forming bacteria to normal.

A decrease in the level of fibrinogen in the blood in comparison with the norm shows that there is an acute infection in the human body, because an increase in its number during postoperative therapy indicates a decrease in the number of infectious pathogens in the body and its recovery. As can be seen from table 91, decamethoxin leads to normal fibrinogen levels faster, which shows its clear advantage over the comparison tool Nifuroxazide.

An increase in prothrombin time indicates that vitamin K and the development of dysbiosis in the intestine are not absorbed in the human body. In this case, it can be seen that decamethoxin improves the condition of the intestine 4 times faster than Nifuroxazide.

As can be seen from the data in table 92, the indicators of the liver and kidney samples of patients are within normal limits, but it is clearly seen that when using Nifuroxazide, the indicators strongly shift to the lower limit of the norm, and when using a solution of decamethoxine, the indicators almost do not change, which shows a significant advantage of using decamethoxin without harm to human metabolism. Table 90

Hematological parameters of patients in conditions

S.aureu infectious coloproctitis

Table 91

Patients hemostasis in conditions

infectious coloproctitis caused by S. aureus

Table 92

Biochemical parameters of blood serum of patients with infectious coloproctitis caused by S. aureus

A study of the second group of patients with treatment without surgical intervention.

The study involved 30 patients. All patients were diagnosed with acute infectious coloproctitis caused by bacteria Staphylococcus aureus.

All patients included in the study were divided into 2 clinical groups (see tables 93-94) for 15 people: patients of group 1 were prescribed Nifuroxazide as therapy, and for patients with GTI 2, 0.02-0.2 mass% solution of decamethoxin was administered orally.

Table 93

Gender distribution of patients

Table 94

The average age of patients in the studied groups

Table 95

From the data in table 95 it is seen that, compared with the initial state, the average body weight of patients during treatment with Nifuroxazide did not change, and when treated with a solution of decamethoxin, the average body weight of the patient increased by 4%.

From the information noted above, it can be seen that decamethoxin affects the course of the disease better than Nifuroxazide: wounds in the intestine are better healed, assimilation of nutrients is established, which leads to a set of patients.

Table 96

From the data in table 96 it can be seen that the decamethoxin solution is better than the comparison tool. Nifuroxazide affects the development of Staphylococcus aureus in the human intestine, reducing the number of coliform bacteria to normal.

From the data of tables 97 and 98 it is seen that Nifuroxazide and decamethoxin do not affect the coagulation time. A decrease in the level of fibrinogen in the blood in comparison with the norm shows that there is an acute infection in the human body, because an increase in its amount during therapy indicates a decrease in the number of infectious pathogens in the body and its recovery. As can be seen from table 98, decamethoxin leads to normal fibrinogen levels faster, which shows its clear advantage over the comparison tool Nifuroxazide. Table 97

Hematological parameters of patients in conditions of 1 infectious coloproctitis caused by S. aureus

Patients hemostasis in conditions of infectious coloproctitis caused by S. aureus

An increase in prothrombin time indicates that vitamin K and the development of dysbiosis in the intestine are not absorbed in the human body. In this case, it can be seen that decamethoxin improves the condition of the intestine 4 times faster for Nifuroxazide. Table 99

As can be seen from the data in table 99, the indicators of the liver and kidney samples of the patients are within normal limits, but it is clearly seen that when using Nifuroxazide, the indicators strongly shift to the lower limit of the norm, and when using the decamethoxin solution, the indicators almost do not change, which shows a significant advantage of using decamethoxin without harm to human metabolism.

From the results of the studies, it is seen that under the conditions of infectious coloproctitis caused by S. aureus, when administered 0.02-0.2 mass% solution of decamethoxin, it exhibits pronounced antimicrobial properties that exceed the antimicrobial activity of Nifuroxazide and contribute to the restoration of the functional state of the patient’s body. So, unlike Nifuroxazide, decamethoxin causes a significant 4% increase in the average body weight of patients compared to the initial state. The pronounced antimicrobial activity of decamethoxin is confirmed by a decrease in the normal number of colony forming units of the pathogen on the 6th day of the experiment, while under the influence of Nifuroxazide this indicator does not reach the norm. According to the severity of the antimicrobial action, decamethoxin exceeds the reference Nifuroxazide by 7.4 times.

The therapeutic effect and antimicrobial effect of decamethoxin is evidenced by a reliable renewal of the leukocyte count indicators: eosinophils, lymphocytes and monocytes, as compared with the action of H | furoxazide. Decamethoxin has a noticeable positive effect on the development of the pathological process in the human colon, helps to clean the surface of the mucous membrane from foci of colonization of microorganisms, prevents the destruction of the surface epithelium, and reduces cell infiltration of the lamina propria mucosa. The therapeutic effect of decamethoxin is superior to the comparison drug "Nifuroxazide" in the treatment of infectious

73

FIXED SHEET (RULE 91) ISAUA coloproctitis caused by S. aureus.

It can also be seen from the above experiments on patients that decamethoxin treats infectious coloproctitis equally well, both when used in postoperative therapy and in the treatment of infectious coloproctitis without surgery, which eliminates the need for surgery and significantly simplifies the treatment of infectious coloproctitis.

The research results are presented and conclusions indicate that decamethoxin can be used orally for the treatment of gastrointestinal diseases, it is practically not absorbed in the gastrointestinal tract, acts only in the intestinal lumen and does not affect the vital functions of other organ systems.

General conclusions regarding the action of decamethcosine when taken orally for the treatment of gastrointestinal diseases:

- With prolonged (28 days) oral administration, decamethocosin does not exert a locally irritating effect on the gastrointestinal mucosa of laboratory animals in a therapeutic dose.

According to the results of the studies, it was found that decamethoxin in the form of a solution exhibits pronounced antimicrobial activity, relative to gram-positive (S. aureus, S. pyogenes, B.subtilis), and gram-negative (E.coli, K. pneumoniae) microorganisms. In relation to P. aeruginosa, antimicrobial activity is less pronounced. Relative to fungi of the genus Candida, decamethoxin showed pronounced fungistatic activity. Thus, decamethoxin exhibits pronounced antimicrobial activity in vitro and can be recommended for the treatment of intestinal infections.

Under the conditions of infectious coloproctitis caused by S. aureus, when administered 0.02 mass% of a solution of decamethoxin, it exhibits pronounced antimicrobial properties that exceed the antimicrobial activity of Nifuroxazide and helps to restore the functional state of the body. The therapeutic effect and antimicrobial action of decamethoxin is evidenced by a reliable renewal of the leukocyte formula indicators: eosinophils, lymphocytes and monocytes, as compared with the control pathology group. Decamethoxin has a significant positive effect on the development of the pathological process in the colon. It helps to clean the surface of the mucous membrane from the foci of colonization of microorganisms, prevents the destruction of the surface epithelium, reduces cell infiltration of the own plate of the mucous membrane. The therapeutic effect of decamethoxin is superior to the comparison drug "Nifuroxazide" in the model of infectious coloproctitis caused by S. Aureus.

In conditions of infectious coloproctitis caused by P. aeruginosa, when administered 0.02-0.2 mass% solutions of decamethoxin, it exhibits pronounced antimicrobial properties that exceed the antimicrobial activity of Nifuroxazide and contributes to the resumption of the functional state of the body.

It was established that a pronounced antimicrobial properties in relation to P. aeruginosa, E. coli, S. aureus, S. paratyphi and C. albicans is inherent in a decamethoxin solution. The severity of the inhibitory effect of a decamethoxin solution depends on the type of microorganism and pH. The antibacterial activity of decamethoxin relative to test strains of bacteria and fungi was enhanced in an alkaline environment.

Decamethoxin in all studied doses (10.0 μg, 25.0 μg, 50.0 μg, 100.0 μg) exhibits pronounced antimicrobial properties. The diameters of growth inhibition zones of test microorganisms vary depending on the type of microorganism and pH. The highest activity of decamethoxin solution in relation to P. aeruginosa, S. aureus, E. coli is observed at pH 8.0 ± 61617; 0.1, relative to C, albicans at 7.15 ± 61617; 0.2, relative to S. paratyphi at pH values of 8.0  0.1 and 5.1  0.2.

A solution of decamethoxin dose-dependently violates the film formation of microorganisms. According to the degree of inhibitory effect with respect to biofilms of P. aeruginosa and E. coli, the activity of the test solution to decamethoxin changed depending on the pH value. The most expressive inhibitory effect of decamethoxin is recorded at a pH of 8.0  0.1 at a concentration of 10.0 MIC.

It was found that the formation of biomass of microorganisms under the action of decamethoxin depends on the concentration of the compound, exposure time and pH of the medium. Inhibition of the growth of E. coli biomass is observed after 1 hour at pH values of 8.0  0.1 and increases with increasing incubation time. A pronounced inhibitory effect against S. paratyphi is observed after 24 and 48 hours in an acidic and alkaline environment. Inhibition of the growth and reproduction of P. aeruginosa is observed starting from 6 hours of decamethoxin action at a pH of 6.3 0,4 0.4 and increases with an increase in the incubation period. S. aureus exhibits a sensitivity to decamethoxin in the first hours of action (after 6 hours) at a pH of 5.1 0,2 0.216. After 24 and 48 hours, the most pronounced inhibition of the growth of Staphylococcus aureus biomass is recorded at a pH of 5.1 & 0.216 and a pH of 6.3 # 0.416. It was found that the pH value practically does not affect the sensitivity of C. albicans fungi to decamethoxin.

Thus, the above convincingly indicates that oral decamethoxin for the treatment of gastrointestinal diseases is a highly effective drug, relatively safe and promising for implementation in medical practice, as a new pharmaceutically active substance for the treatment of intestinal infections of microbial and viral etiology. Decamethoxin is also a new pharmaceutically active substance for the treatment of infectious coloproctitis without surgery and for improving the treatment of infectious coloproctitis in surgery.

75

FIXED SHEET (RULE 91) ISAUA

Claims

FORMULA

The use of decamethoscine as a pharmaceutically active substance for the treatment of diseases of the gastrointestinal tract and intestinal infections orally.

The use of decamethoxin according to claim 1, characterized in that decamethoxin is used to treat diseases of the gastrointestinal tract and intestinal infections of microbial and viral etiology.

The use of decamethoxin according to paragraph 1, characterized in that decamethoxin is used to treat infectious coloproctitis.

The use of decamethoxin according to paragraph 1, characterized in that decamethoxin is used in the form of an aqueous solution with a concentration of decamethoxin 0.02-0.2 mass%.

The use of decamethoxin according to paragraph 1, characterized in that decamethoxin is used as part of the pharmaceutical composition.

The use of decamethoxin according to paragraph 1, characterized in that decamethoxin is used as part of a pharmaceutical composition of a liquid type.