RU2048807C1 - Antibacterial and antiinflammatory preparation - Google Patents

Abstract

FIELD: veterinary. SUBSTANCE: proposed preparation contains (mass): sodium formiate 0.5-44.7, sodium acetate 34.8-43.6, sodium propionate 6.3-36.7, sodium butyrate 5.1-2.8. EFFECT: improves quality of desired product. 9 tbl

Description

 The invention relates to the production of medicines for veterinary medicine.

 It is known that young animals of domestic animals and birds are sensitive to intestinal infection, leading to the occurrence of enterocolitis, diarrhea and, as a consequence, a decrease in safety and growth in the early neonatal period, when there is a high energy dependence of the body due to the lack of a well-functioning thermoregulation system. In particular, this is characteristic of piglets and chickens. Hypersensitivity of sucking piglets to feed antibodies is also noted.

 Known drugs belonging to the class of antibiotics of a number of tetracyclines, for example, biomycin, which is widely used for the treatment and prevention of enterocolitis in piglets and broiler chickens.

 The disadvantages of these drugs include numerous side effects associated with lesions of the mucous membranes, suppression of local immunity, factors of non-specific resistance, the occurrence of candidiasis.

 Drugs are known that belong to the class of aminoglycosides, for example, colimycin, used in the treatment of gastroenteritis in piglets. The disadvantages of this drug include high oto-, nephrotoxicity, provoking dysbiosis.

 Known drugs of the nitrofuran series used in the treatment of salmonellosis in piglets, in particular furazolidone. The disadvantages of this class of drugs include their lack of effectiveness, hepatotoxicity, and frequent allergic reactions.

Antimicrobials are known (mainly in the form of a lotion or gel) containing salts of C ₅ -C ₁₂ fatty acids. The disadvantage of these drugs is that they are xenobiotics, have a relatively narrow spectrum of antimicrobial activity and low biological activity.

 The aim of the invention is to increase therapeutic efficacy, in combination with an antibacterial effect, normalization of the autoflora infrastructure and an increase in mucosal resistance.

This goal is achieved by an antibacterial anti-inflammatory drug, which is a mixture of sodium salts of short-chain monocarboxylic acids in the following ratios of components, wt. Sodium formate 0.5-44.7 Sodium acetate 34.8-43.6 Sodium propionate 6.3-36.7 Sodium butyrate 5.1-28.0
Checking the claimed technical solution for compliance with its criterion, the inventive step showed that neither in the scientific nor in the patent literature revealed a combination of features specified in the characterizing part of the claims. The composition is a set of metabolites of saccharolytic anaerobic animal autoflora, but in this case obtained chemically.

The composition is obtained by neutralizing a previously distilled mixture of formic, acetic, propionic and butyric acids, sodium hydroxide. An equimolar amount of sodium hydroxide in water (50% solution) is added to the aqueous solution of acids taken in certain mass fractions. After short stirring, the solvent is removed in vacuo and dry the residue is dried to constant weight at 140 ^C. The composition of the process is reproducible when the boundary and the averaged values of the initial components.

 The following is the technology for the preparation of example 4.

PRI me R. 401 g of formic acid, 440 g of acetic acid, 102 g of propionic acid and 61 g of butyric acid are charged into a 5 liter glass container equipped with a stirrer and a coil refrigerator. Then, with a rotating mixer for 15-20 minutes add 724 g of sodium hydroxide in the form of an aqueous 50% solution. Thus the solution temperature rises to ^about 70-85 C. After stirring the solution was transferred into an evaporator during heating and is removed in vacuo half volume of water. The hot solution was placed in a vacuum drying cabinet, wherein at 110-120 ^{° C} and a pressure of 2-5 mmHg within 3-5 hours, the solvent residue is removed and the product is brought to constant weight. The resulting white residue is crushed and ground. The product contains a mixture of formate, acetate, propionate and sodium butyrate in mass fractions of 42.8, 43.2, 8.9, 5.1, respectively.

 Similarly receive the compositions shown in examples NN 1-12.

 The drug is a white or light yellow powder, readily soluble in water, physiological saline and aqueous solution of sodium chloride. The drug can be stored in plastic packaging at room temperature for 2 years.

Acute and chronic toxicity of the composition was studied in C ₅₇ Bl / 6 mice and Wistar rats. The drug was administered intraperitoneally at doses of 6-300 mg / kg (i.e., in the therapeutic and a 20 and 30 and a 50-fold excess). It was found that the composition with the exception of example No. 5 does not affect the general condition of animals and their behavioral reactions.

A pathomorphological study of internal organs and an analysis of serum biochemical parameters confirmed the absence of pathogenic effects. There is an increase in body weight. The LD ₅₀ for mice was 4980 mg / kg, for rats 4432 mg / kg. For the composition according to example 5, 1866 and 1521 mg / kg were obtained, respectively, and therefore this composition was not included in the protected range. With oral administration, it was found that the drug does not show signs of local irritant effect (lack of hyperemia, edema, ulceration and necrosis of the mucosa).

 A study was also conducted of the specific toxicity of the composition, in particular, its mutagenic activity, embryotoxicity, and the study of allergenic properties.

 The study of mutagenic activity was carried out by methods of accounting for recessive, sex-linked, lethal mutations in Dr. melanogaster and dominant lethal mutations in germ cells of mice. It was found that the composition when administered orally in a dose range of 6-2490 mg / kg does not have mutagenic properties.

 The exception was the composition according to example 9, which found a significant increased proportion of chromosomes with flying recessive mutations (0.58 ± 0.18 at 0.19 ± 0.12 in the control). In connection with the revealed circumstance, the composition of N 9 is not included in the protected range. In the study of allergenic activity, reactions of active skin anaphylaxis, systemic anaphylaxis, immune complexes, delayed-type hypersensitivity and mast cell degranulation were studied. Information obtained during a systematic assessment of the allergenic properties of the composition indicates the absence of an allergenic effect in the studied doses of 6-60 mg / kg. It was also found that the composition lacks embryotoxic and teratogenic properties.

 The following are examples of formulations with antibacterial and anti-inflammatory properties (examples 1-4) and examples of formulations that go beyond the protected range.

 PRI me R 1.

Component, wt. Sodium formate 0.5 Sodium acetate 34.8 Sodium propionate 36.7 Sodium butyrate 28.0
PRI me R 2.

Component, wt. Sodium formate 44.7 Sodium acetate 43.6 Sodium propionate 6.3 Sodium butyrate 5.4
PRI me R 3.

Component, wt. Sodium formate 22.4 Sodium acetate 39.2 Sodium propionate 24.7 Sodium butyrate 13.7
PRI me R 4.

Component, wt. Sodium formate 42.8 Sodium acetate 43.2 Sodium propionate 8.9 Sodium butyrate 5.1
PRI me R 5.

Component, wt. Sodium formate 45.0 Sodium acetate 38.8 Sodium propionate 10.6 Sodium butyrate 5.6
PRI me R 6.

Component, wt. Sodium formate 0.4 Sodium acetate 42.0 Sodium propionate 36.0 Sodium butyrate 21.6
PRI me R 7.

Component, wt. Sodium formate 39.2 Sodium acetate 34.0 Sodium propionate 16.5 Sodium butyrate 10.3
PRI me R 8.

Component, wt. Sodium formate 21.1 Sodium acetate 45.0 Sodium propionate 26.4 Sodium butyrate 7.5
PRI me R 9.

Component, wt. Sodium formate 1.9 Sodium acetate 35.6 Sodium propionate 33.5 Sodium butyrate 29.0
PRI me R 10.

Component, wt. Sodium formate 28.2 Sodium acetate 36.7 Sodium propionate 30.6 Sodium butyrate 4.5
PRI me R 11.

Component, wt. Sodium formate 20.2 Sodium acetate 36.1 Sodium propionate 38.0 Sodium butyrate 5.7
PRI me R 12.

Component, wt. Sodium formate 43.6 Sodium acetate 42.9 Sodium propionate 6.0 Sodium butyrate 7.5
Salmonellosis (paratyphoid) is one of the widespread infectious diseases of weaned piglets. Caused by the paratyphoid group bacteria Salmonella [1b] In this regard, the bacteriostatic effect of the composition in vitro was evaluated by its effect on Salmonella.

(1)
Из данных табл.1 видно, что составы 2, 4, 5 с высоким содержанием формиата натрия обладают довольно значительным ингибирующим эффектом по отношению к S. enteritidis, у состава 4 с высоким содержанием формиата и пропионата эффект максимален. Состав 1, не содержащий формиата натрия, сравнительно менее эффективен, а состав, с содержанием формиата, выходящий за пределы диапазона, даже стимулирует рост сальмонелл (N 6). В связи с этим, состав по примеру 6 не включен в защищаемый диапазон.S.enteritidis culture was grown for 18 hours at 37 ^{° C} in tubes with 10 ml broth (Todd-Hewitt) to a density of inoculum 1˙10 ⁹ CFU / ml in the presence of the test composition ( "experience") or without ( "control" ) In this case, the drug is taken in such a dose that the final concentration corresponds to the compositions of the examples. Evaluation of the effects was carried out by measuring the optical density (OD) of bacterial suspensions on a Perkin-Elmer spectrophotometer in a standard cuvette at a wavelength of 450 nm. The percentage of inhibition is calculated by the formula:
Ing.

(1)
From the data of Table 1 it can be seen that compositions 2, 4, 5 with a high content of sodium formate have a rather significant inhibitory effect with respect to S. enteritidis, while in composition 4 with a high content of formate and propionate the effect is maximal. Composition 1 that does not contain sodium formate is relatively less effective, and a composition with a formate content outside the range even stimulates the growth of salmonella (N 6). In this regard, the composition of example 6 is not included in the protected range.

 The bacteriostatic effect of the composition is also evaluated by its effect on the pathogenic β-hemolytic streptococcus group A (Str. Pyogenes), which is known to be the etiological agent of various diseases of the respiratory, gastrointestinal tract, skin diseases, systemic arthritis, etc. and in humans and animals. In this regard, the bacteriostatic effect of the composition was evaluated by its effect on museum and natural strains of streptococcus gr. A and gr. V.

 A culture of S-pyogenes type-M29 in matt form (strain D33 / 11 N 62/59, Prague collection) was grown similarly to the previous example. In the same way, growth inhibition was evaluated.

 From the data of table 2 it is seen that the effectiveness of inhibition is largely determined by the proportion of propionate in the composition. In particular, when the content of this component is less than 6.3%, a sharp decrease in the inhibitory effect occurs in the composition (Example 12). The composition of example 12 is not included in the protected range.

 Hospital strain Str. agalactiae (Str.B) was grown as described above, and the inhibitory effect of the drug was similarly studied.

 Examples 1,4 show that various variants of the composition have the ability to inhibit the growth of streptococcus B in culture. Moreover, the composition containing formate is more effective (Table 3).

 The antimicrobial activity of the composition was evaluated by studying its effect on the virulence factors of typical pathogenic Str. pyogenes and non-pathogenic streptococcus Str. salivarius, respectively. The effect of the composition on hydrophobicity and adhesive properties was studied.

The hydrophobicity of the culture is estimated by spectrophotometric study of the optical density of cell suspensions at a wavelength of 450 nm (Perkin-Elmer spectrophotometer). Examine the 18-hour culture of Streptococcus grown in Todd-Hewitt broth at 37 ° ^C. The hydrophobicity of the culture is measured by the difference of the optical densities of the cell suspensions, referred to the initial (%) in 0.01 M sodium phosphate buffer with 0.15 M NaCl before and after shaking with hexadecane, at 6000-7000 d for 1 min on Vortex'e.

 From the data of table 4 it is seen that the composition according to examples 1, 4 reduces the initial hydrophobicity of pathogenic streptococcus. The hydrophobicity of Str. pyogenes is determined mainly by the content and exposure in the cell wall of one of the main factors of virulence, the M protein. The result can be considered as evidence of a decrease in the virulence of the pathogenic culture.

 In contrast, the composition of Example 8, when exposed to a culture of S. pyogenes, exhibits the ability to increase the hydrophobicity of pathogenic streptococcus cells. The composition of example 8 is not included in the protected range.

 Exposure to non-pathogenic S. salivarius leads to a slight increase in its hydrophobicity, i.e. adhesion ability.

×100% где IА_k индекс адгезии в растворе;
IА_о индекс адгезии в присутствии состава.A decrease in the adhesive properties of β-hemolytic streptococcus is also manifested in model experiments on suspensions of piglet buccal epithelium. Cells from the inner surface of the cheeks are scraped off with a wooden spatula, placed in buffered saline solution (PBS) at a pH of 7.2-7.4, and washed from indigenous microflora by centrifugation at 1000 g. The washed epithelium is incubated for 10 min at 87 ^about with a culture of Salmonella enteritidis, in the presence or without antibacterial composition. Then the epithelial cells are washed and after fixation and staining are examined under a light microscope. Visually highlight the proportion of infected cells, the average number of bacteria per cell (x), the adhesion index (1A), equal to the product of the proportion of infected cells by x, as well as the adhesion efficiency indicator by the formula
E ₁ =

× 100% where IA _{k is} the adhesion index in solution;
IA _on the adhesion index in the presence of the composition.

 Thus, the compositions noticeably prevent the adhesion of pathogenic streptococcus to a suspension of buccal epithelium, excluding the composition of example 8, the effectiveness of which is noticeably lower in this indicator (Table 4).

The antimicrobial activity of the composition is also evaluated in vivo in C57 Black mice infected by intraperitoneal administration of a suspension of pathogenic streptococcus (Table 6). Use a dose of Str. pyogenes equal to LD ₅₀ (0.5-1.0 x 10 ⁷ kOU / ml) in a volume of 0.5 ml. Twenty minutes after the administration of the streptococcus suspension, the mice of the experimental group were also injected intraperitoneally with 0.5 ml of physiological saline at a dose of 40 mg. Mice in the control group are injected with 0.5 ml of saline. At the end of the experiment, surviving animals are sacrificed and pathological changes in internal organs are evaluated.

 Three groups of piglets were formed, three nests (10 suckers) in each. One group served as control. Pigs of the first experimental group are fed the composition according to example 1, the second group according to example 4, the third according to example 10.

 In different nests of each of the three groups, different administration schemes were used. In one of the nests, the drug was fed orally from 6 days of age, in the other two from the beginning of feeding, the results were averaged. The general feeding schedule was standard. The dose of the drug in all cases was the same 1.5 g of the composition in 10 ml of water daily. Duration of administration is 20 days. The dynamics of the live weight of sucking piglets is presented in table.7.

 Thus, sucking pigs receiving a preparation at a dose of 577-91 mg / kg in the pre-weaning period show a noticeable increase.

 A colonoscopic examination was performed with a biopsy of piglets from all 4 groups, one from each group. The endoscopic picture of representatives of 3 experimental groups without features, crypt hyperplasia, smearing of the vascular pattern and some neutrophilic infiltration of lamina propria were noted in the control group. These signs can be considered as a manifestation of local hypersensitivity to complementary antigens characteristic of early complementary feeding [3] Since this reaction is controlled by secretory immunoglobulin A (S-IgA), its amount in the intestinal contents was studied using the Mancini method using a diagnostic kit for secretory immunoglobulins produced by Biomed. The determination results are shown in table 8, in terms of dynamics.

 Immediately after weaning, all the piglets in the control group developed diarrhea. Enteropathogenic E. coli strains were found in crops. Colonoscopic examination showed a significant increase in neutrophilic mucosal infiltration. Other signs of inflammation were noted, in particular, mucosal hyperemia and crypt hyperplasia.

 Analysis of secretory s-IgA in intestinal contents showed that inflammation leads to a decrease in the proportion of s-IgA compared to IgA.

 On the contrary, diarrhea did not occur in the experimental groups, a cytological examination did not reveal neutrophilic infiltration, and the [lgAl / [s-lgA] ratio remained less than 1, hyperemic phenomena disappeared, the vascular pattern returned to normal, crypt hyperplasia was practically not observed (Table 8).

 Thus, the composition exhibits the ability to enhance local immunity through the development of protective immunoglobulins equipped with a protective glycoprotein, as well as anti-inflammatory activity, preventing the "illegal" infiltration of the mucous membrane and its own plastic by neutrophils, contributing to the elimination of endoscopic signs of inflammation.

 The exception is the composition according to example 10, in which this ability is expressed to a lesser extent, in particular, in the group 6 cases of diarrhea were detected. The composition is not included in the protected range.

 The use of pigments in the control group and the group of Example 10 for the diarrhea-infected pigments of Examples 1 and 4 in doses of 1.5 g / day, respectively, allowed the animals to be cured within 3 days.

 At the Krasnogorskaya poultry farm, the influence of the compositions according to examples 2, 3, 7, 11 on the safety and weight gain of young chickens was studied. 8 groups of birds were formed (4 experimental and 4 control) of 200 animals each with the same number of males and layers. The compositions were introduced daily into the standard diet PK-4M of young poultry in the first 10 days of life immediately upon receipt from a hatchery at a dose of 50 mg per head. T.O. the initial dose was about 2.5 g / kg.

 The results of the experiment are presented in table.9. From the data of the table it can be seen that the compositions according to examples 2 and 3 significantly increase the gain in males, but to a lesser extent in young females. The composition according to example 2 significantly increases the safety of juveniles, the composition of example 3, on the contrary, to a greater extent affects the safety of males. The reason for the departure in most cases was colibacteriosis. In the whole group, both compounds provide a significant (33-35%) increase in biomass. The compositions according to examples 7 and 11 generally do not provide a cumulative effect on the group, and a tendency to a decrease in safety among youngsters is revealed. In this regard, both compositions were not included in the protected range.

 Thus, studies of the composition showed that it has antibacterial activity in relation to both gram-positive and gram-negative microflora, inhibits its adhesion to epithelium, it also has an anti-inflammatory effect, and enhances local immunity.

 The composition has neither general nor specific toxicity, has no side effects on the body of experimental animals, sucking pigs, young birds, as it is a combination of natural metabolites normally produced by saccharolytic anaerobes. In the framework of this technical solution, this set of metabolites is obtained chemically.

 The composition is highly soluble in water, convenient for transportation, stable for 2 years, can be introduced into the body of an animal in the form of a solution, tablets, capsules and can be used both with a filler and directly.

Claims (1)

 ANTIBACTERIAL ANTI-INFLAMMATORY PRODUCT containing fatty acid salts, characterized in that it contains sodium formate, sodium acetate, sodium propionate and sodium butyrate in the following ratio of components, wt. Sodium formate 0.5 44.7
Sodium Acetate 34.8 43.6
Sodium Propionate 6.3 36.7
Sodium butyrate 2.8 5.1

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