RU2840855C1 - Antimicrobial agent with wound healing effect for treating infectious diseases of distal extremities of cattle - Google Patents

Abstract

FIELD: veterinary science.

SUBSTANCE: invention refers to veterinary science and may be used for treating bovine distal extremities diseases. Wound healing antimicrobial agent in the form of a gel for treating infectious diseases of bovine distal extremities contains the following ingredients, wt.%: copper disodium ethylenediaminetetraacetate chelate 4.16, zinc disodium ethylenediaminetetraacetate chelate 2.72, formic acid 0.21, acetic acid 0.11, lactic acid 0.17, carboxymethyl cellulose 3, distilled water – balance.

EFFECT: disclosed antimicrobial agent has pronounced antimicrobial properties and wound healing effect, high therapeutic effectiveness in treating finger and toe diseases of infectious and non-infectious aetiology in the initial and moderate severity, has no toxic effect on the animal's body.

1 cl, 2 tbl, 2 ex

Description

The invention relates to the field of veterinary medicine and can be used to treat diseases of the distal extremities of cattle due to its pronounced antimicrobial and wound-healing properties.

In recent decades, diseases of the fingers and hooves have caused the greatest damage to dairy cattle breeding. Due to the physiological characteristics of highly productive cows, frequent production violations during their use and failure to provide timely veterinary and zootechnical care for injuries, various lesions of the distal limbs of non-infectious (injuries, aseptic laminitis and pododermatitis, ulcers of the pulp, coronary band and vault of the interdigital cleft) and infectious (necrobacteriosis, digital dermatitis and other purulent-necrotic and ulcerative infections) etiology have become widespread [1, 2]. A large number of local and systemic agents have been proposed and tested for non-specific prevention and treatment of cattle with diseases of the distal limbs [3-5]. However, their use and effectiveness are limited by the need for significant financial and labor costs, the complexity of implementation in the technological scheme of dairy cattle breeding and restrictions on milk. In addition, in most cases these remedies are used with a great delay. As a result, surgical manipulations with the use of expensive local (external, local) remedies and symptomatic therapy are required, so the effectiveness of the treatment is minimal.

When creating effective preparations for external use, carriers of active and auxiliary substances are biodegradable polymeric substances - gels, powders, sponges, films. Despite a fairly wide choice of dosage forms used for the prevention and treatment of superficial and deep lesions of the extremities, the choice of a gel as a carrier of active and auxiliary substances of a drug is the most preferable, since gels form reliable mechanical protection and actively resist infection, which has been convincingly proven in practice by the long-term and effective use of Solka Hoofgel®, containing chelated copper and zinc compounds [6].

Damage to the fingers and hooves is caused by a large group of opportunistic microorganisms, which do not cause any diseases in healthy animals under comfortable conditions. The pathogenic effect of these microorganisms manifests itself when they penetrate into another ecological niche, a sharp decrease in immunobiological resistance and exceeding the infectious threshold of the body [7]. One of the main pathogens of acute purulent infections is staphylococcus, which actively destroys the protective properties of the skin with its enzyme systems during maceration and microtrauma, opening the "gates" for other microorganisms. Usually, microorganisms that have entered the wound require 6-12 hours to adapt and actively manifest their vital activity. Therefore, it is important to maintain mechanical protection of the skin in a timely manner [8, 9].

Gel-based preparations, preventing the adhesion of microorganisms to tissues, reduce the concentration of vegetative and spore forms of bacteria, fungi and viruses in the wound area, preventing the development of infection.

In diseases of the skin of the hoof horn and underlying tissues of ungulates, the best results were shown by imported gel preparations, but in recent years they have become less accessible to veterinary practice, and the arsenal of domestic preparations of this kind with sufficient effectiveness is presented on the market in small quantities. Therefore, the development of effective domestic remedies for the treatment of diseases of the distal extremities in a short time is very relevant.

The prior art discloses the drug necrogel, which includes medicinal substances from the groups of antibiotics, sulfonamides, local anesthetics, antiseptics and excipients, for the treatment of purulent-necrotic processes in the area of the distal parts of the extremities of cattle and the method for its production (patent RU 2204997, IPC A61K 31/00, published 27.05.2003, bulletin No. 15). The drug has an antimicrobial effect on microorganisms involved in the infectious purulent-necrotic process in necrobacteriosis of animals. The disadvantage is that it includes antibiotics, which limits the use of this drug in lactating cows due to the waiting period for milk.

The drug Tricillin (manufacturer: OOO NPK Askont+, Russia) is known from the state of the art - a powder for the prevention and treatment of necrobacteriosis and foot rot of the distal limbs and hooves in cows. The active ingredients are benzylpenicillin, streptomycin and sulfanilamide. Among the disadvantages, one can highlight the bacteriostatic rather than bactericidal effect on gram-positive, gram-negative, aerobic and anaerobic bacteria, cocci, spirochetes and mycobacteria, as well as the fact that slaughter of animals for meat is allowed 6 days after the cessation of the use of Tricillin. The meat of animals forced to be killed before the expiration of the specified period is used to feed carnivores or to produce meat and bone meal. The disadvantages include the fact that milk obtained during treatment and within 6 days after the last administration of Tricillin is prohibited from being used for food purposes; such milk can be used in animal feed after boiling.

The state of the art includes the drug Chemi spray (Industrial Veternaria S.A. Invesa, Spain), which is used to treat skin and hoof lesions caused by necrobacteriosis. The drug has pronounced antibacterial and anti-inflammatory properties. The active substance is an antibiotic of the tetracycline group: chlortetracycline hydrochloride and gentian violet. The negative side of using the drug is the possibility of developing antibiotic resistance and the antibiotic getting into milk.

A drug is known for the treatment and prevention of skin and integument diseases of infectious etiology in cattle, small cattle, and pigs based on copper chelates (see patent RU 2706694, IPC A61K 31/30, A61K 33/34, Published: 20.11.2019 Bulletin No. 32), containing the following components in the following proportions, %: copper chelate - 20-30, lentex M4 - 30-40, lentex B352R - 30-40, dye - 0.01-0.05. This drug is used in the form of a gel. The disadvantages include the lack of comprehensive treatment.

The closest in technology to the claimed product is the Solka Hoofgel® hoof gel (Kanters Special Products B.V., Netherlands), which contains minerals in the form of chelated copper and zinc compounds and organic acids, and is taken as a prototype. A significant disadvantage of this product is its complex composition, high cost and limited availability of purchase in the Russian Federation.

Unlike Solka Hoofgel®, the claimed product, while maintaining the effectiveness at the prototype level, contains only domestic components, and its use is cost-effective.

A significant novelty of the claimed agent is also the proposed method for synthesizing the chelates included in its composition, with the transition through the hydroxy form of metal ions in an aqueous solution, which differs from the prototype in that it allows for the chelation reaction to be carried out quickly and with a completeness of up to 100%, forming stable coordination and chemical bonds with a monodentate ligand. This form contributes to the good penetrating ability of the complex with high transport functions for the delivery of metal ions (copper and zinc) to the area of inflammatory/damaged tissue, causing a high antimicrobial and wound-healing therapeutic effect.

The objective of the proposed invention is to develop an antibiotic-free, inexpensive, effective and easy-to-use product based on chelated copper and zinc compounds, which has pronounced antimicrobial properties and a wound-healing effect, and which allows for obtaining products (milk, meat, offal) that can be used without restrictions.

The task is solved by creating an antimicrobial agent with a wound-healing effect for the treatment of infectious diseases of the distal extremities of cattle, which contains the following components, wt. %:

disodium ethylenediaminetetraacetate copper chelate - 4.16;

zinc disodium ethylenediaminetetraacetate chelate - 2.72;

formic acid - 0.21;

acetic acid - 0.11;

lactic acid - 0.17;

carboxymethylcellulose – 3;

distilled water - the rest.

The claimed product is in the form of a gel. The gel base facilitates the penetration of the active components of the product: sodium ethylenediaminetetraacetate copper chelate and sodium ethylenediaminetetraacetate zinc chelate - deep into the tissues, where they have a pronounced antimicrobial, anti-inflammatory and wound-healing effect. And the supporting and attaching components - carboxymethylcellulose and organic acids - facilitate long-term adhesion of the product to the affected areas, preventing their contamination.

A comparative analysis of the claimed solution with known means used for the treatment of infectious diseases of the distal extremities of cattle allows us to conclude that it meets the criterion of “novelty”, since as a result of the conducted patent information search, the proposed set of essential features of the invention was not found.

Comparison of the claimed technical solution with those known from the technical field allowed us to identify features that are not obvious to a specialist in connection with the technical result achieved by the entire set of essential features. This allows us to conclude that the claimed technical solution complies with the criterion of "inventive step".

The claimed technical solution meets the criterion of “industrial applicability”, as it can be used to treat diseases of the distal extremities of cattle in production conditions - agricultural enterprises of various levels.

The method for preparing an antimicrobial agent with a wound-healing effect for the treatment of diseases of the distal extremities of cattle is as follows

Among the complexing metals, copper (II) has been studied particularly intensively [10, 11]. This readily available and very common metal in the oxidation state +2 forms a huge number of complex compounds of various compositions and structures, including mono-, bi-, tri-, tetranuclear complexes and other polynuclear compounds, including polymeric, heterovalent and heterometallic compounds. Some classes of copper (II) complex compounds and the features of their structure and magnetic properties are summarized in recent reviews [12, 13].

The main objective of obtaining a gel-like substance was to obtain supertransportable copper and zinc ions with high penetrating ability and diffusion into tissue cells. The simplest solution in this case was to obtain simple stable complexonates capable of transporting these ions. Stable complexes were formed on the basis of ethylenediaminetetraacetic acid derivatives (NaEDTA, Trilon B). The obtained complexes were converted into a gel-like state in a carboxymethylcellulose solution. Only chemically pure grade substances were used. To prepare 1 liter (1 kg) of gel, distilled water was used as a solvent. Chelate solutions are prepared separately.

Copper ions were isolated in pure form from the solution as copper hydroxide precipitates. A sample of 41.66 grams of copper sulfate crystal hydrate (CuSO ₄ *5H ₂ O) was taken, dissolved in 400 ml of water, and an alkali solution of 15 grams of crystalline sodium hydroxide (NaOH) was added with a slight excess of 50 ml of water. The resulting sodium sulfate completely leaves with the aqueous portion of the mixture upon three-fold washing of the precipitates with short periods of settling of the heterogeneous mixture (approximately 5-10 minutes). Precipitates freshly isolated by decantation and three-fold washing with distilled water provide an advantage over stale substances introduced as dispersed crystalline powders, since gel-like hydroxide precipitate has a more developed surface for interaction and obtaining complex forms of chelates ("claw-shaped") with completeness of flow and formation of stable coordination bonds with the amino group and chemical bond by the carboxyl group. There is practically no unreacted part of the substances. Blue copper hydroxide gel completely dissolves when heated, forming a blue transparent solution. The amounts of substances are taken in a stoichiometric ratio of 1:1 complexing ion with Na-EDTA, for each ion, 50 grams of complexone were taken.

A similar mechanism of interaction occurs with zinc ions. A white freshly formed precipitate of zinc hydroxide, prepared from 27.16 grams of crystalline zinc sulfate, 400 ml of water and 50 ml of water with 15 grams of sodium hydroxide dissolved in it, becomes completely transparent when interacting with a solution of Na-EDTA, giving a stable chelate.

To speed up the reaction, the solutions were heated to boiling. In addition, to prepare gels of effective consistency, it is necessary to concentrate evaporate the solutions by approximately 1.5 times, i.e. we bring the initial volumes from 450 to 300 ml of each solution of copper and zinc chelates.

After the solutions have cooled, add a gelling agent - crystalline Na-carboxymethylcellulose at a rate of 3-5% of the solution weight, for the desired solution consistency from 30 to 50 grams. Mix thoroughly and leave the mixture to soak for about 30 minutes, after which mix the resulting gels again.

As an additional agent, we introduce concentrated formic acid 1.2% by weight - 2.14 grams; acetic acid 0.65% by weight - 1.08 grams and lactic acid 1.05% by weight - 1.75 grams into the gels. After thorough mixing and homogenization, we combine the copper and zinc gel solutions, transfer them to a sealed container and put them in a cool place.

The finished product is packaged in polymer jars with a capacity of 300; 500; 1000 g according to GOST 33756.

The effectiveness of the use of the claimed antimicrobial agent can be illustrated by the following examples.

Example 1. Evaluation of the therapeutic efficacy of the claimed product in the form of a gel for the treatment of diseases of the distal extremities of cattle at the Chepchugi branch of Biryuli Moloko LLC.

In the production experiment conducted at the Chepchugi department of Biryuli Moloko LLC, groups of animals with different stages and clinical manifestations of finger and claw diseases were formed. For work, a machine with a split for fixing animals was installed in a technologically convenient place, where examination, cleaning, trimming of hooves, sanitation and treatment of finger and claw diseases were carried out. After the procedure of trimming hooves, removing dirt, dead tissue, pus, irrigating the wound with a 3% solution of hydrogen peroxide, 1% solution of chlorhexidine, or 1.0% solution of furacilin and subsequent tamponade with dry gauze napkins, the declared agent was applied to the affected area with a brush or spatula in a dose of 5 to 20 g, depending on the affected area. After all manipulations, the animal was left in a fixed position for 10-15 minutes. If necessary, the wound area was protected with a light bandage dressing; repeated treatments were carried out at intervals of 3-5 days.

Before, during and after treatment, the number of sick animals was recorded and the degree of limb damage was assessed. The results of bacteriological studies of biological material samples indicated the presence of a wide variety of bacteria in smears from the lesions of the limbs: F. necrophorum, Streptococcus spp., Staphylococcus spp., Clostridium spp. and microscopic fungi (Aspergillus spp., Penicillium spp. and Mucor spp., Candida albicans).

Diseases of infectious and non-infectious etiology were diagnosed based on characteristic clinical signs and laboratory studies of biological material samples with the isolation of pathogenic cultures of microorganisms.

The course of treatment for cows in each group was carried out according to the following scheme: day 1 - treatment; day 3 - examination, treatment as needed; day 5 - examination, treatment as needed; day 7 - examination and evaluation of the treatment result.

The treatment results are presented in Table 1.

Possessing pronounced antimicrobial and wound-healing properties, the claimed product prevented the development of inflammatory, purulent-necrotic and ulcerative processes. When treating a mild (initial) degree of damage to skin tissue and hoof horn, a single application of the claimed product to the wound surface was sufficient to stop the inflammatory phenomena and infection. The average degree of damage required 2-3 treatments.

After using the claimed product, the improvement in the clinical condition of the animals and the reduction in wound healing time correlated with the severity of the clinical manifestation of the disease and the reduction in the number and species composition of microorganisms in smears-prints in samples of biological material from the affected fingers. Thus, if before the use of the drug in cows with limb diseases among a large variety of opportunistic microorganisms detected, the number of one or more of their representatives (F. necrohorum, Streptococcus spp., Staphylococcus spp., Clostridium spp., Aspergillus spp., Penicillium spp., Mucor spp. and Candida albicans) exceeded the threshold value (more than 1×10 ⁴ CFU), then after the use of the declared agent in cows with mild and moderate severity of the disease, vegetative forms of bacteria and microscopic fungi were completely absent, and among animals with a severe degree, spore and anaerobic bacteria continued to be detected.

Thus, the claimed remedy has shown high therapeutic efficiency in the treatment of diseases of fingers and hooves of infectious and non-infectious etiology in the initial and moderate severity of lesions. The expediency of prolonged treatment of severe lesions was determined up to 7 days of treatment, with treatment up to 5 times. However, the probability of a favorable outcome of such lesions, with a high probability of relapses of the disease, was no more than 30-45%, which is confirmed by the data of other researchers.

Example 2. Evaluation of the therapeutic efficacy of the claimed product for the treatment of cattle hoof diseases in comparison with the prototype Solka Hoofgel®.

The therapeutic efficacy of the claimed product was studied in comparison with the commercial imported product Solka Hoofgel® (Kanters Special Products B.V., Netherlands), taken as a prototype, in a strictly controlled experiment on 23 animals with mild to moderate manifestations of finger and hoof diseases, which were divided into five groups.

The affected areas of the limbs of sick animals after “rigid” fixation, cleaning, trimming of the deformed horn of the hooves and removal of necrotic tissues were treated with the declared agent or prototype, keeping the fixed limb (5-10 minutes) until the gel dried.

In the first group: 5 heads with an initial degree of damage, after wound sanitation: cleaning, trimming of hooves and excision of necrotic tissue, the declared product was applied without applying a bandage. In the second: 5 heads with an average degree of damage, after cleaning, trimming of hooves and excision of necrotic tissue, the wound was applied with the declared product and covered with a bandage. The third group: 5 heads with an initial degree of damage, treated with Solka Hoofgel® without a bandage. In the fourth group: 5 heads with an average degree of damage, after wound sanitation, Solka Hoofgel® was applied and covered with a bandage. The fifth control group: 3 heads, not treated.

In case of mild damage to fingers and hooves, the inflammatory process in animals after application of the declared product to cows in the first experimental group (5 heads) and the prototype in the third group (5 heads) stopped on the 3rd day.

During the treatment period, the bandage and gauze dressing were removed after 72 hours in animals with moderate finger damage. If necessary, the wound surface was sanitized with antiseptic treatment and the preparations were reapplied. The duration of treatment depended on the severity of the lesion and the rate of tissue healing. As a result, the inflammatory process completely stopped in all cows in the experimental groups on the 5th and 7th day of observation. The use of the claimed remedy for the treatment of diseases of the distal limbs in cows, along with the prototype, contributed to the cessation of the smell of decay, the fastest relief of inflammatory processes, and the disappearance of lameness after 2-3 procedures.

Thus, in cows of the experimental groups, purulent-necrotic processes in the area of the fingers and hooves ceased, and in the control group (3 heads) deep necrosis of the finger tissues was noted in the form of purulent pododermatitis, phlegmon or abscess.

With favorable regenerative processes and no additional injuries, complete recovery of the cows in the experimental groups occurred on the 10th-15th day. As a rule, no new cases of the disease were noted in the experimental groups during subsequent observation periods. During orthopedic examination of the cows in the experimental groups on the 15th day after the use of drugs, the condition of the soft tissues and hoof horn was without obvious defects and pathologies.

The results of selective bacteriological studies of the microflora in the area of the distal extremities of treated cows indicated a decrease in the microbial count and the absence of most types of microorganisms found in pathological material during the disease, which had a positive effect on regenerative processes and restoration of the functions of the fingers and hooves in cows (Table 2).

As a result of the research, it was established that when applied externally, the claimed product is harmless and has no side effects. At the same time, the claimed product is not inferior in effectiveness to the commercial imported drug Solka Hoofgel®, which has proven itself on the market, and, on par with it, quickly stops the inflammatory process, accelerating the resorption of the infiltrate and stimulating regenerative processes in the tissues of the fingers and hooves.

The use of the claimed product in agricultural enterprises with unfavorable conditions for diseases of fingers and hooves has shown its high therapeutic effectiveness in such diseases as: necrobacteriosis, digital dermatitis, ulcers of the pulp, coronary band, interdigital cleft vault, as well as traumatic purulent pododermatitis, abscesses and phlegmons in the finger area.

Example 3. Study of the therapeutic efficacy of the claimed product for infectious diseases of the fingers and hooves of cattle in OOO SKHP Elgan in Uninsky District of Kirov Region

During the clinical examination of 800 cows in two barns of the Yelgan Agricultural Farm, 200 lame animals were identified. After the orthopedic medical examination and forced culling of seriously ill animals, 100 cows with mild and 51 cows with moderate damage to the toes and hooves remained in the premises.

The effectiveness of the claimed product was assessed in a strictly controlled experiment on 36 cows. Animals with mild to moderate manifestations of finger and hoof diseases were divided into two groups based on the principle of analogues.

The claimed product was evenly distributed on the affected toes and hooves of the cows of the first group (18 heads) after wound sanitation (cleaning, trimming of hooves and excision of necrotic tissues) followed by the application of a protective gauze bandage. The lesions of the cows of the second (control) group (18 heads) after wound sanitation were treated with Chemi spray and a gauze bandage was also applied.

In case of mild damage to fingers and hooves, the inflammatory process in animals after application of the declared product to cows in the experimental group (8 heads) and Chemi spray in the control group (9 heads) stopped on the 3rd day.

During the treatment period, the gauze bandage was removed after 72 hours in animals with moderate toe damage. If necessary, the wound surface was sanitized with antiseptic treatment and the preparations were reapplied. The duration of treatment depended on the severity of the damage and the rate of tissue healing. As a result, all cows in the experimental and control groups recovered. The recovery of the experimental group cows (10 heads) occurred on the 10th-15th day, and the control group (9 heads) on the 13th-18th day.

As a result of the research, it was established that the claimed product is harmless when applied externally, has no side effects and is not inferior in effectiveness to the drug Chemi spray. At the same time, the claimed product stops the inflammatory process faster, accelerating the resorption of the infiltrate and stimulating regenerative processes in the tissues of the fingers and hooves.

Thus, the results of preliminary tests of the declared product in a number of agricultural enterprises of the Republic of Tatarstan, Nizhny Novgorod and Kirov regions, unfavorable for diseases of the fingers and hooves of cattle, indicate the high efficiency of this product and the possibility of its wide implementation in the system of measures to combat diseases of the fingers and hooves of cattle.

List of references

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Claims (8)

An antimicrobial agent with a wound-healing effect for the treatment of infectious diseases of the distal extremities of cattle, containing the following components in the following ratio, wt. %: disodium ethylenediaminetetraacetate copper chelate - 4.16; zinc disodium ethylenediaminetetraacetate chelate - 2.72; formic acid - 0.21; acetic acid - 0.11; lactic acid - 0.17; carboxymethylcellulose - 3.00; distilled water - the rest.