Method of obtaining complex antibacterial immunomodulating preparation

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the chemical-pharmaceutical industry and represents a method of obtaining a complex preparation for the application in veterinary, possessing immunomodulating and antiseptic properties, which includes mixing succinic acid, levamisole and formalin in distilled water with the following component ratio, wt %: levamisole - 3.0%-3.5%; succinic acid - 2.0%-2.5%; formalin - 0.3%-0.5%, distilled water - the remaining part; sterilisation of the solution by autoclaving is performed in a mode of 1 atm for 20 minutes.

EFFECT: elaboration of the method of obtaining the complex preparation for the application in veterinary, possessing immunomodulating and antiseptic properties.

 

The invention relates to veterinary medicine and relates to methods of production of complex drugs with immunomodulatory and antiseptic properties.

A method of obtaining drug "amber biostimulator" to increase the resistance of the organism of animals (RF patent No. 2303979, 2007), which makes it possible to obtain biologically active drug in aqueous solution, including its composition 1% succinic acid, 4% antiseptic stimulant Dorogov second fraction (ASD-2), 0.25% novocaine, pH of 6.9-7.0 and. The method of obtaining comprehensive drug increases the body's resistance of animals to adverse environmental factors, normalizes metabolic processes in the cells in various immune deficiencies, metabolic disorders, Exo - and endogenous intoxication. However, the drug does not have sufficient antibacterial effect.

A method of obtaining a comprehensive immunotropic antiseptic drug for the treatment and prevention of infectious diseases in animals, having in its composition in addition to the above formalin has antiseptic properties (patent RF №2361579, 2009), but it does not have a strong immunomodulatory effect.

The task of the invention to provide injectable form of the drug with a broad spectrum Biol which policy actions with purposeful action on the cellular immune system and antibacterial properties.

This object is achieved by the inclusion in the integrated product of succinic acid, levamisole and formalin in the following ratio of components, wt.%:

Levamisole - 3,0%is 3.5%;

Succinic acid and 2.0%to 2.5%;

Formalin - 0.3%and 0.5%;

Distilled water - the rest.

An important feature of the invention is the presence of levamisole, which is a highly effective immunostimulant, predominantly cellular immune system.

In the study of the biological action of levamisole was found that it increases the overall resistance of the organism and can be used as a tool for immunotherapy. Experiments on isolated cells and the monitoring of healthy and ill patients showed that the drug can restore the changed functions of T-lymphocytes and phagocytes and because of his demominations effect can regulate cellular mechanisms of the immune system. More detailed studies have shown that levamisole, selectively stimulating regulatory function of T-lymphocytes, can function as an immunomodulator that can strengthen weak cellular immune responses, to weaken strong and not to act normal.

In connection with these properties levamisole has been proposed for the treatment of various diseases, the pathogenesis of which the value of disorders of immunogenesis: primary and secondary immunodeficiency, autoimmune disease, chronic and recurring infections, tumors and other

However, levamisole has a strong side, mainly convulsive effect on the body, however, there is a method of reducing the side effects of levamisole due to the inclusion of part of the preparation of succinic acid (patent RF №2411944, 2011).

In the integrated drug, in addition to succinic acid and levamisole, as an antibacterial component includes vitamin formalin.

The inclusion of formalin in the integrated drug due to its exceptionally high antiseptic activity. So, in a concentration of 1:6000 it stops the growth of the typhoid bacilli, and absolutely low concentration of 1:30000 stops the rot broth (I.E. Brain Pharmacology. Moscow - "Kolos", 1979, s).

It is known that intramuscular (parenteral) administration of formalin at a concentration of 0.1-0.2% allows to reduce the virulence of the pathogen virus transmissible gastroenteritis of pigs directly in the body of animals and at the same time to stimulate specific immune protection (Laskawy NR. Method of prevention of transmissible gastroenteritis of swine. RF patent №2028804 from 20.02.1995).

An example of the wasp is estline way

For preparation of the comprehensive drug used 940,0 ml of distilled water, in which successively dissolved 20,0 g of succinic acid, which corresponded to 2% concentration; 30.0 g of levamisole. Add 10 ml of formalin (30%) allows to obtain an optimal environment for the action of levamisole in the pH range of 4.5-5.0,

The received complex immunometabolic product is a clear, sterile solution. Qualitative and quantitative composition the proposed drug in comparison with a standard solution of levamisole and the prototype can markedly reduce the toxicity of anthelmintica-immunostimulant of levamisole, to provide high hepatoprotective and immunometabolic effect. Qualitative and quantitative composition the proposed drug provides a synergetic effect, which is illustrated by examples of its experimental tests.

Determination of the optimal concentration of formalin held on the results of study of the safety and bacterial activity prototypes of the integrated product. With this purpose were experimental samples preparation containing 0.2%; 0,3%; 0,4%and 0.5% formalin.

Bactericidal activity was determined in the experiment in vitro, standard method of test delay microbial growth around the paper disks, capitanich the above solutions integrated product. As a test object used culture of Staphylococcus aureus, planted in bacteriological Cup mastopathy agar. After 24-hour exposure in thermostat at 37°C, the diameters of zones of growth inhibition of microorganisms was around disks impregnated complex preparation containing formalin 0,2% - 5-7 mm; 0.3% to 10-12 mm, 0.4% of 15-17 mm; 0,5% - 20-22 mm Diameter zones delay 15 mm and more showed high bactericidal activity.

Test harmlessness spent on white mice. Daily for 5 days intraperitoneal injection in a volume of 0.5 ml samples of the drug containing 0.2%, 0.3% and 0.4% formalin, was not accompanied by the suppression of the General condition experimental animals. The introduction of the drug containing 0.5% formalin for 5 days resulted in reduced activity of the animals, however, death was not observed.

Thus, the results of pilot experiments determined that to ensure the antiseptic activity of the integrated product is the most optimal concentration of formalin is not less than 0.3%, but not above 0.5%.

Study of antibacterial activity

Experience spent on cows with clinical signs of mastitis at the end of the lactation period. Experimental animals were divided according to the principle analogues into two groups. Cows of the first group (n=3) 7 pain is diversified shares of the breast nutrizionale in an amount of 5 ml was injected the drug, containing 0.4% formalin. Animals of the second group (n=3) with 5 patients shares nutrizionale was introduced amber biostimulant (RF patent No. 2303979) in the same volume. The control antibacterial activity of drugs in the treatment of mastitis spent a day of their introduction and after 5 and 10 days after injection. The results of the experiment are shown in table 1.

Research results indicate that the inclusion of the drug formalin resulted in an accelerated process of neutralization microflora in patients with mastitis lobes of the breast.

The study of the immunostimulatory activity of the claimed preparation on pigs, Sosunov

For the experiment were selected from two groups of sows, three heads each, in which the choke was 10 goals piglets.

In 4-day-old pigs from the experimental group (n=30) was intramuscularly injected with the test drug in the dose of 1.5 ml, and the pigs of the control group (n=30) Seferovic. Control studies and their results are shown in table 2.

When comparing results of studies conducted on the 7th day, with background indicators found that piglets from the experimental and control groups revealed some differences. Thus, the amount of erythrocytes in piglets of the experimental group increased by 5%, 4% increased saturation aritri the tov hemoglobin. The indicator of reserve alkalinity increased to the lower limit of the physiological norm, which indicates that the elimination of nutritional disorders. This is due to the fact that succinic acid in combination with levamisole is the most effective drug in the elimination of nutritional disorders. The improvement of acid-alkaline balance of the blood pointed to the normalization of disturbed metabolism. Especially pronounced changes occurred in the protein and mineral metabolism. The level of protein and blood levels of total calcium in piglets of the experimental group on day 7 has reached the physiological norm. The level of lymphocytes in the blood of piglets from the experimental group on the 7th day of 7.4% was more than individuals from the control group that showed improvement of their immune status. This is also pointed out trend growth BASK and FAL.

Studying the activity of cellular immunity in calves-dairy

For the experiment two groups were formed calves analogues in live weight and age parameters. The control group calves were injected with standard solution of levamisole 7.5% in the dose of 2.5 ml, experimental group - tested version of the drug at a dose of 2.5 ml

The results obtained (table 3) allow you to mark that on the 20th day, the number of red blood cells in calves of the experimental group was higher by 12.4% (p<0.05)than in animals of the control group, gematik is it at 10 days of age was higher by 9.7% (p< 0,05). This tends to increase and the level of hemoglobin in the blood of the calves of the experimental group.

In calves from the experimental group before the first feeding of colostrum number of leukocytes was by 24.2% (p<0.05), and 30 days of age 14.6% (p<0,05) higher than in animals of the control group, indicating that the activation of cellular immunity factors, however, there is a positive tendency to increase hemoglobin in the blood of the calves.

The results of research on the effectiveness of the drug received showed that the drug has a strong antibacterial effect and effectively acts on the immune system of animals that can effectively treat infectious diseases and immunodeficiency animals of various etiologies.

Table 1. Antimicrobial effectiveness of the proposed drug and amber biostimulator in the treatment of clinical mastitis in cows

CowMedicationThe species composition of the microflora before and after drug injection
Before the introduction ofHa 5 dayHa 10 day
The claimed product with 0.3% formali is and
1.E. coli Str. agalactiae-
2.S. aureus Str. agalactiaeS. aureus-
3.S. aureus Str. agalactiae-
Amber biostimulant
1.E. Coli Str. agalactiaeStr. agalactiae-
2.S. aureus Str. agalactiaeS. aureus Str. agalactiae-
3.S. aureus E. coliS. aureus-

Table 2. The effect of the drug on hematological and immunobiochemical the blood of piglets, n=10

IndicatorsPeriods of research, d
Background indicators7142128
Erythrocytes, 1012/l3,8±0,23,8±0,24,0±0,23,8±0,24,1±0,23,9±0,24,4±0,33,9±0,24,5±0,34,0±0,2
Hemoglobin, g/l78,56±2,8479,42±3,26 81,37±2,9479,68±2,7582,47±3,2578,46±2,6788,72±3,5679,24±2,2789,24±4,3280,27±2,78
Protein, g/l52,3±2,253,7±2,454,8±3,153,5±2,657,2±3,453,2±3,658,6±3,254,1 ±3,159,3±3,754,8±3,6
Total calcium, mmol/l1,44±0,101,48±0,181,56±0,121,53±0,111,98±0,171,55±0,162,02±0,141,57±0,182,05±0,121,59±0,13
Inorganic phosphorus, mmol1,32±0,161,31±0,12/math> 1,34±0,121,33±0,141,36±0,181,33±0,111,41±0,121,36±0,151,45±0,141,38±0,12
Alkalinity,% vol. CO235,1±1,635,3±1,734,2±2,135,2±1,339,4±3,2*35,4±1,5 39,5±3,1*35,6±1,739,8±2,6*35,8±1,9
Leukocytes,
109/l
7,22±0,367,46±0,28of 7.48±0,427,32±0,447,56±0,847,29±0,327,92±0,527,26±0,228,02±0,757,24±0,23
Lymphocytes, %48,18±3,4 49,72±2,98EUR 54.15±3,7450,12±2,8459,85±of 3.4651,26±2,4859,73±4,1152,23±2,3661,48±4,3353,18±2,75
BASK, %39,61±2,740,32±2,648,83±2,941,23±2,253,64±3,242,34±2,6 53,81±2,947,21±2,756,44±3,2848,63±2,80
FAL, %21,8±2,522,3±2,425,4±2,721,8±2,529,3±2,522,0±2,530,1±2,722,6±2,432,3±3,122,8±2,9
Note: the top row is the performance of piglets from the experimental group; the bottom line is the performance p is reset control group; p<0,05-0,01*

Table 3. The effect of the drug on hematological indices of newborn calves, n=10

IndicatorsAge, days
Before feeding colostrum102030
Erythrocytes, 1012/lof 7.93±0,62 7,76±0,517,80±0,63 7,37±0,378,75±0,53* to 7.67±0,368,10±0,64 7,50±0,37
Hemoglobin, g/l116,4±6,75 116,6±4,0of 114.5±6,1 110,1±of 4.38118,9±4,25 byr111.4±6,46for 96.1±6,16 92,0±3,52
Hematocrit, %40,0±2,42 37,1±1,7138,4±1,94* 34,7±1,4837,3±3,01 39,7±1,7831,5±2,31 29,7±1,43
Leukocytes, 109/l10,9±0,43* of 8.28±0,78of 8.40±0,59 8,3 5±0,59was 9.33±1,2 9,10±0,758,07±0,59* 6,89±0,41
Stab, % 1,83±0,31 1,90±0,31---
---
Segment-nuclear, %56,8±4,03 was 59.9±1,8444,0±8,15 47,7±a 4.8333,5±4,96 42,0±4,2431,5±4,74 27,3±1,89
Monocytes, %4,43±0,89 3,30±0,74to 4.41±1,53 4,10±0,498,81±2,28 of 6.61±0,736,05±1,51* 3,63±0,51
Lymphocytes, %37,4±3,95 35,3±1,9950,6±7,13 41,b±equal to 4.9757,0±to 5.21 of 48.7±4,5362,0±3,27 64,8±3,16
Note: the top row is the performance of the experimental group; the bottom line is the performance of the control group; * p<0,05-0,01

The method of obtaining comprehensive antibacterial, immunomodulatory agent, including mixing in distilled water succinic acid, formalin, sterilization by autoclaving, characterized in that its composition is optionally administered levamisole in the following ratio of components, wt.%:
Levamisole - 3,0%is 3.5%;
Succinic acid is - of 2.0%to 2.5%;
Formalin - 0.3%and 0.5%;
Distilled water - the rest,
and sterilization by autoclaving is carried out in mode 1 ATM for 20 minutes.



 

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