RU2640482C2 - Supramolecular complex of triclabendazole for treatment of animals in case of fascioliasis - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: method comprises enteral administration of a supramolecular complex of triclabendazole and arabinogalactan in a ratio of 1:10 in the form of a powder of 0.1 to 10 microns in size, where the animals are received the complex in the form of an aqueous solution or in a dry mixture with food at a dose of 1-2 mg/kg.

EFFECT: increased anthelmintic efficacy.

4 ex, 4 tbl

Description

The invention relates to veterinary medicine, in particular to helminthology, and can be used for the prevention and treatment of animals with acute and chronic form of fascioliasis.

Among the most common and dangerous parasitic diseases of animals and humans is fascioliasis, which is widespread in the world and in Russia. The disease proceeds both acutely and chronically, often with a severe clinical course.

Fascioliasis causes significant economic damage to livestock. Fasciols, parasitizing in the liver, cause severe pathological changes, especially during the acute course of the disease.

Thus, in case of fascioliasis, the economic damage consists of culling the liver, reducing milk productivity by 10-15%, weight gain of young animals by 5-20%, slaughter yield of beef by 3.28%, shearing of wool of sheep by 10-18%, meat quality by 100-300 kcal / kg and the death of animals, especially sheep, in the acute form of the disease.

As a result of the analysis of available data in recent years, the average infection rate of cattle and small cattle with fascioli in Russia is 28.6%, and in some of its regions it reaches up to 90.0% (AM Ataev, (1990) [2]; Arkhipov I.A. ., 2004 [1]; EE Kolyada, (2004) [6]; V.V. Loshkareva, (2005) [4]; V.V. Gorokhov, (2012) [7] and others [3 , 5, 7, 8, 9].

For deworming of animals during fascioliasis, a large number of foreign anthelmintics have been proposed: rafoxanide, oxyclosanide, closantel and domestic dosage forms based on them. However, these drugs are not effective against young fasciol.

The only veterinary and medical practice active against young fasciols is the imported drug Siba-Geigi, triclabendazole (fazinex-5% suspension), which has been widely used abroad.

The closest technical solution, selected as a prototype, is the substance Triclabendazole. Synonyms: (CGA 89317, fazineks). The drug is more effective against young fasciol, however, the effectiveness of triclabendazole with fasciol age decreases slightly. The therapeutic dose for triclabendazole for cattle is 12 mg / kg, and for sheep and goats 10 mg / kg. The drug is administered orally with food or individually in the form of a suspension once [1, 4].

The disadvantage of the selected prototype is its high cost, it is practically insoluble in water and up to 70.0% are excreted from the body into the external environment unchanged with feces, polluting the environment.

Given the enormous economic damage caused by fascioliasis, the development of effective drugs is highly relevant. Developed earlier in VNIIP them. K.I. Scriabin preparations are polytrem against chronic and atsamidofen against the acute form of fascioliasis are not currently produced, and imported triclabendazole (fazineks) in Russia is practically not used due to the high cost.

In this regard, the goal of our work was to obtain a domestic preparation in the form of a supramolecular complex based on the substance triclabendazole (TKB) and water-soluble polymers using modern nanotechnologies to increase the solubility, bioavailability of triclabendazole, and increase anthelmintic and economic efficiency.

The proposed anthelmintic agent (called “Triclafascid” by us) is obtained using mechanochemical technology in impact-grinding type grinders with adjustable energy intensity. As a water-soluble polysaccharide, arabinogalactan (AH) is used, isolated from larch, which is an environmentally friendly and safe product that is widely used in medicine and veterinary medicine.

Obtaining a supramolecular complex with triclabendazole includes the following processes:

1) Weighing the components (substance TKB and polysaccharide AG) in a weight ratio of 1:10 and pre-mixing in a drum mounted on the rolls of the mill LE-101 for 10-15 minutes;

2) Loading grinding media into the drum and activation of the mixture of components for 2 hours while rotating the drum at a speed of 90 rpm to obtain the drug triclafascid;

3) Unloading of the preparation and packing in polyethylene containers with tightly closing lids of 100-500 g.

The resulting preparation Triclafascid is a guest-host supramolecular complex in the form of a loose powder ranging in size from 0.1-10 microns, light beige in color with a light coniferous smell and a sweetish taste. Triclafascid, unlike the substance triclabendazole, is highly soluble in water, more convenient to use and has the following advantages:

1) The supramolecular complex is obtained in one stage without the participation of liquid phases in one stage, in contrast to the previously developed method (using acetone or ethyl alcohol [11]). It should be noted that this eliminates the process of dissolution, precipitation, drying of the product; no liquid and solid waste.

2) The cost of treating an animal with Triclafascid is reduced by 8 times compared with the substance triclabendazole, due to a decrease in the therapeutic dose.

It is known that water-insoluble anthelmintics used by animals orally, up to 70.0% are excreted into the environment unchanged with feces, polluting the environment. The water-soluble supramolecular complex Triclafascid, used in a dose reduced by 8 times, absorbed into the blood, provides high bioavailability to parasites. In the future, the drug is split and excreted mainly with urine from the body in scanty amounts, without polluting the environment.

The above positive signs of the obtained supramolecular complex of Triclafascide compared with the prototype of the substance triclabendazole ensure that the technical solution meets the criterion of "novelty" and this allows us to conclude that the technical solution meets the criterion of "significant differences".

Examples of specific performance.

Example 1. Obtaining supramolecular complexes based on triclabendazole using different pharmacopoeial polymers.

In order to obtain the most optimal dosage form with high anthelmintic and economic effectiveness, we have developed 10 innovative drugs in the form of supramolecular complexes based on the substance triclabendazole with various water-soluble polymers:

1) Triclabendazole / hydroxyethyl starch (TKB / HES) = 1/10;

2) Triclabendazole / polyvinylpyrrolidone (TKB / PVP) = 1/10;

3) Triclabendazole / arabinogalactan / pharmacopeia (TKB / AGF) = 1/10;

4) Triclabendazole / arabinogalactan / technical (TKB / AGT) = 1/10;

5) Triclabendazole / sodium salt of carboxymethyl cellulose (TKB / Na CMC) = 1/2;

6) Triclabendazole / silica grade (volcasil) (TKB / SiO ₂ ) = 1/2;

7) Triclabendazole / Fucus (TKB / F) = 1/4 and 1/10;

8) Triclabendazole / kelp (TKB / L) = 1/4 and 1/10.

Example 2. Obtaining suspensions of triclabendazole with polymers.

Suspension forms of triclabendazole were also obtained in a roller mill using various auxiliary components (surfactants, structure-forming agents, etc.) and polymers. The compositions of the resulting suspensions are shown below:

1) Triclabendazole / polyvinylpyrrolidone, 10% suspension of TKB based on PVP;

2) Triclabendazole / arabinogalactan, 10% suspension. TKB based on AG.

Example 3. Titration of the obtained supramolecular samples of TKB with polymer fillers according to the parameters of acute toxicity.

One of the important parameters developed and introduced into practice of drugs is the determination of their toxicity.

In this regard, we conducted toxicological screening of the drugs proposed in the framework of this application.

Before a pet test, a check is required that includes the shortest tests that carry the necessary information. In this regard, to determine the parameters of acute toxicity, we tested the above-mentioned samples of TKB with various polymer excipients on 400 white outbred mice weighing 18-20 g. The drugs were administered orally into the stomach using a special probe in doses from 4000 to 20,000 mg / kg. Each dose of the drug was tested in 10 animals.

The parameters of acute toxicity in white mice were determined by the Lichfield and Wilcoxon method in the Roth modification (ML Belenky, 1963) [5, 9], taking into account the mortality of animals from the administered doses of the studied drugs with the definition of indicators: LD ₀ ; LD ₁₆ ; LD ₅₀ ; LD ₈₄ ; LD ₁₀₀ . Assessment of the degree of danger of the preparations was carried out according to GOST 12.1.007-76.

According to the results of the experiments, it was found that all 10 TKB samples with different polymer excipients at the enteral route of administration to white mice according to GOST 12.1.007-76 belong to class IV safe substances, of which TKB / AG / T had the least toxicity (the results are shown in the table No. 1, appendix).

Example 4. Testing the effectiveness specified in example 1 and 2 samples of supramolecular complexes of triclabendazole in fascioliasis of sheep.

Experience No. 1 Testing the substance of triclabendazole and its 5 supramolecular complexes was carried out in the Southern Federal District on 67 sheep of the Tushino breed spontaneously invaded by fascioli.

To determine the degree of invasion of animals, feces were taken individually and examined using the Fulleborn method to determine the number of fasciolus eggs on average g / feces. Sheep spontaneously invaded by fascioli were selected in the experiment and were distributed according to the principle of analogues to 6 experimental animals of 10 (n = 10) sheep and the control (n = 7) group. The animals of the first experimental group received the basic preparation, the substance triclabendazole, at a dose of 10.0 mg / kg by DV in the form of an aqueous suspension. The second - TKB - 10% suspension based on PVP, the third - TKB / PVP = 1/10, the fourth - TKB / SiO ₂ = 1/10, the fifth - TKB / HES = 1/10, the sixth - TKB / NaKMTS = 1 / 10. All these drugs are well suspended in water and are soluble powders. The seventh group of sheep received a 10% suspension. The drugs were individually administered once at a dose of 10 mg / kg powder or 1.0 mg / kg according to the active substance (DW), in the form of aqueous suspensions. The eighth group of sheep did not receive the drug, as it served as a control. After giving the drugs for 3 days, the animals underwent clinical observation.

To determine the effectiveness, 25 days after the drug was given, feces were taken individually from the experimental and control animals and investigated similarly to the selection of animals in the experiment.

The effectiveness of triclabendazole dosage forms was monitored according to the type of “control test” according to the Guidelines approved by the World Association for the Progress of Veterinary Parasitology (1995) [10].

The test results of the complexes of triclabendazole with polymers are summarized in table No. 2, which shows that the basic drug triclabendazole at a dose of 10 mg / kg in DV released 9 out of 10 animals, the extensivity (EE) was 90.0%, and the egg intensity was fasciol in g / feces decreased (IE) by 99.0%. A 10.0% suspension of TKB based on PVP at a dose of 1.0 mg / kg in terms of DV produced an EE of 90.0%, IE = 97.4%. The remaining preparations showed the following results: the TKB / PVP complex = 1/10 showed EE = 90.0% and IE = 97.5%; TKB / SiO ₂ = 1/10 - EE = 80.0%, IE = 94.5%; TKB / HES = 1/10 - EE = 70.0%, IE = 95.4%; TKB / NaKMTs = 1/10 - EE = 70.0%, IE = 87.9%. In animals of the control group, the number of eggs in g / feces averaged 104.7 ± 7.8 specimens.

Thus, the supramolecular complexes of TKB showed high efficacy against fasciol in doses 10 times less compared to the base drug. In the process of work, it was noted that the TKB / HEC = 1/10 complex at high atmospheric humidity and temperatures above + 30 ° C is caking, and for the TKB / NaKMTs = 1/2 complex, time is required for polymer swelling, which increases the time spent on mass deworming individually, in an aqueous solution of TKB / SiO ₂ = 1/2 there is a slight precipitate of silicon dioxide.

All tested samples of triclabendazole supramolecular complexes by animals were well tolerated; we did not note any deviations from the physiological norm and side effects.

Experience No. 2. A test of the effectiveness of the proposed supramolecular complexes TKB / AGT = 1/10, TKB / AGF = 1: 10, TKB / L = 1/10 TKB / F = 1: 10 was carried out on 58 sheep of the Tushino breed, spontaneously invaded by fascioli. Sheep selected in the experiment were distributed according to the principle of analogues to 5 experimental animals (n = 10-12) in the group. The animals of the first experimental group received TKB / AGT = 1/10, the second - TKB / AGF = 1: 10, the third - TKB / F = 1: 10, the fourth - TKB / L = 1/10. All of the above drugs are fine powders that easily form suspensions when mixed with water. All four samples were injected at a dose of 10 mg / kg according to the preparation, and at a dose of 1.0 mg / kg in an aqueous solution in a DV, the fifth group of sheep received a basic preparation of TKB at a dose of 1.0 mg / kg in a DV, as a control. Drugs were given individually orally once from a plastic bottle.

Accounting for the effectiveness of triclabendazole complexes was carried out similarly to experiment No. 1.

The results of testing the effectiveness of the substance triclabendazole and its complexes are summarized in table No. 3, which shows that the basic drug triclabendazole at a dose of 1.0 mg / kg (a 10 times reduced therapeutic dose) was not effective - 27.9%.

The TKB / AGT complex = 1: 10 freed 11 of 12 animals from fasciol; the extensivity in this case is 91.6%, and the intensity of fasciolus eggs in g / feces is reduced by 96.6%. Similarly, other TKB complexes showed the following results: TKB / AGF = 1/10 - EE = 91.6% and IE = 96.5%; TKB / F = 1: 10 - EE = 83.3%, IE = 93.9%; TKB / L = 1/10 - EE = 91.6%, IE = 96.6%.

It should be noted that the tested complexes of triclabendazole showed greater efficacy against fasciol compared with the base drug. In the process of work, it was noted that TKB / F = 1/10 and TKB / L = 1/10 when mixed with water form dense gel-like masses, with which it is difficult to work. Therefore, it is better to give them in a dry mixture with food.

All tested dosage forms of triclabendazole by animals were well tolerated, we did not observe deviations from the physiological norm and side effects.

Summing up the trials of triclabendazole supramolecular complexes, it was found that all samples according to the parameters of acute toxicity at the enteric route of administration to white mice according to GOST 12.1.007-76 belong to class IV safe substances.

A test of the effectiveness of triclabendazole supramolecular complexes showed their rather high extensivity (70.0-100%) on sheep spontaneously invaded by fascioli at a dose of 1.0 mg / kg in DV, which is 10 times lower than the therapeutic norm of the triclabendazole substance itself.

Of the developed and tested samples of the drug, it was necessary to select one sample for further testing in order to introduce it into veterinary practice, which would meet the following parameters, such as anthelmintic and economic efficiency, transportation, storage, solubility in water; smell, taste, photosensitivity, availability for the acquisition of domestic polymer component.

For all of the above parameters, we selected the supramolecular complex of triclabendazole based on the domestic polysaccharide arabinogalactan-technical, conditionally called by us "Triclafascid".

Experience No. 3. Titration of the therapeutic dose of the supramolecular complex Triclafascid.

For titration of the therapeutic dose of the triclabendazole supramolecular complex based on the Triclafascid AGT, 40 Tushino sheep spontaneously invaded by fascioli were selected, which were divided into 4 experimental groups of 10 (n = 10) animals each. The first group of sheep received Triclofascid at a dose of 10 mg / kg according to the preparation (at 1.0 mg / kg DV), the second - 15 (1.5 mg / kg at DV), the third - 20 mg / kg (at 2.0 DV mg / kg), the fourth group of sheep received the basic drug substance triclabendazole at a therapeutic dose of 10 mg / kg for control. The drugs were administered individually orally, once in the form of an aqueous solution.

During the experiment, after giving the drugs, the animals were in the same conditions of keeping and feeding, they were followed up by clinical observations.

The efficiency was taken into account 25 days after the administration of the drugs according to the data of coprooscopic studies using the “critical test” method.

The results of the effectiveness of the drugs are summarized in table No. 4, from which it follows that Triclafascid at a dose of 20 mg / kg for the drug (for DV 2.0 mg / kg) showed 100% effectiveness against mature fasciol, this is an 8 times reduced dose in compared with the substance triclabendazole at a therapeutic dose of 10 mg / kg.

Thus, based on our experience, the smallest dose of the Triclafascid supramolecular complex, at which 100% efficiency is obtained, is 2.0 mg / kg in terms of DV (20 mg / kg in the preparation), and we have established it as a therapeutic for chronic form of fascioliasis of sheep.

Experience No. 4. Testing the effectiveness of the supramolecular complex Triclafascid against preimaginal (immature) fasciol.

In order to determine the effectiveness of the drug on immature forms of fasciol, we used a method for evaluating the effectiveness of the tested drugs with the determination of the content of fasciol eggs in the feces of experimental animals during treatment and a pronounced increase in the egg content after the start of treatment and their subsequent disappearance in the feces of treated animals, revealing the effectiveness of the drug.

This method is based on the phenomenon of early and marked increase in the concentration of helminth eggs in the feces of invasive animals under the influence of effective anthelmintic drugs. Animals invaded by fasciols, but not reaching puberty, in the feces, fasciol eggs are not registered at the beginning, and after treatment, as a result of the decomposition of fascioli under the influence of the drug, an increase in the concentration of unformed gray eggs appears and their subsequent disappearance in feces, we can assume that the animal was cured , and the test drug is considered effective.

To do this, 76 young animals of the first year of grazing were selected for experiment on pasture unfavorable for fascioliasis, in which no fasciol eggs were found in feces, but presumably invaded by fascioli, which were dewormed with the supramolecular complex of triclafascid at a dose of 1.0 mg / kg DV.

The effectiveness of the drug was determined according to coprooscopy, after 3; 7; 14 and 25 days after giving the drug.

As a result of evaluating the effectiveness of the drug by a pronounced increase in the content of fasciol eggs after the start of treatment compared with the initial indicator and the subsequent disappearance of eggs in the feces of the treated animals, it was found that 76 animals of the first year of grazing, which were free from fasciol eggs according to coprooscopy, after giving The drug Triclafascid at a dose of 1.0 mg / kg in 30 of them on the 3rd day unripe eggs (gray) fasciol were detected on average g / fec. 266.0 copies On the 7th - 158.0 copies; 14 - 0.93 specimens., On the 25th day, no fasciol eggs were found.

Therefore, Triclafascid at a dose of 1.0 mg / kg showed a higher 100% effectiveness against preimaginal (immature) fasciol.

According to clinical signs, we did not observe any side effects after giving the drug: body temperature, pulse, respiration and chewing gum were within normal limits, animals were active in movements, took food and water.

A simple economic calculation shows a rather high efficiency of TKB / AG / T due to a reduction in the therapeutic dose by 8 times, since 10 kg of TKB / AG / T = 1/10 can be prepared from 1 kg of TKB substance.

If 1 kg of TKB substance in a therapeutic dose of 10 mg / kg can be processed per sheep with an average live weight of 40 kg, 2500 animals, then 10 kg of TKB / AG / T obtained from 1 kg of TKB substance in a therapeutic dose of 2 mg / kg - 12,500 animals.

As a result, TKB / AG / T can process 10,000 more sheep than TKB substance. The cost of processing one sheep with the substance TKB is 38 rubles, and TKB / AG / T - 7.60 rubles, that is, 5 times cheaper.

Conclusion

By mechanochemical treatment (in the shredders of the abrasion-resistant type) of the substance triclabendazole with polymers, 10 supramolecular complexes of the guest-host type were obtained. The complexes are finely dispersed, free-flowing powders with a particle size of up to 10 microns.

According to the results of titration according to the parameters of acute toxicity, it was found that all complexes with the enteric route of administration to white mice according to GOST 12.1.007-76 belong to class IV low toxic substances, of which TKB / AG / T had less toxicity.

All tested supramolecular samples of triclabendazole versus sexually mature were 8 and immature fasciol of sheep 9 times more effective than the substance of the basic preparation TKB.

For technical and economic reasons, the TKB / AGT complex = 1/10 (Triclafascid), which is promising for implementation in veterinary practice, was selected as the most optimal drug. This sample is also supported by the fact that arabinogalactan, as a well-known biologically active polysaccharide, is used to increase bioavailability, cleanses the body, normalizes the digestive tract, boosts immunity in immunodeficiencies, maintains a normal balance of the microflora of the digestive tract, and is a breeding ground for beneficial bacteria, extremely important for protecting the gastric mucosa from pathogenic organisms. Fasciols, localized in the bile ducts, cause their thickening (calcification), fatty degeneration and cirrhosis of the liver, and arabinogalactan has noticeable hepatoprotective and membranotropic properties, it can be used as a therapeutic agent for the treatment of the liver.

Thus, the claimed anthelmintic agent “Supramolecular complex of triclabendazole with technical arabinogalactan” has good solubility, 8 times more effective than the basic drug triclabendazole, while increasing economic efficiency.

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Claims (1)

A method of treating acute or chronic forms of fascioliasis in sheep, comprising enteral administration of the supramolecular complex of triclabendazole and arabinogalactan in a ratio of 1:10 in the form of a powder ranging in size from 0.1 -10 microns, where the animals are administered the complex in the form of an aqueous solution or in a dry mixture with food in dose of 1-2 mg / kg.