RU2473334C2 - Anti-eimeriosis pharmaceutical composition based on salt of quarternary phosphonium and substituted dinitrobenzofuroxane - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of veterinary. Composition includes n-tetradecyl(tri-n-butyl)phosphonium-chloride and 5,7-bis-(m-nitroanilino)-4,6-dinitrobenzofuroxane as active components and glucose as auxiliary substance with weight ratio of active components 1:10.

EFFECT: composition is not toxic, possesses high therapeutic efficiency.

1 tbl, 3 ex

Description

The invention relates to pharmacy, in particular to antiparasitic pharmaceutical compositions, and can be used as an anti-emeriosis drug in veterinary medicine.

Coccidioses are acute, subacute, and chronically occurring diseases of animals and humans caused by various protozoan species belonging to the Protozoa subdomain, Apicomlexa type, Sporozoa class, Coccidiida order, Eimeriidae family.

Eimeriosis is caused by protozoa that are parasitic mainly in the intestinal epithelial cells. Some species parasitize in the bile ducts of the liver in rabbits (E. stiedae) and the mucous membrane of the geese renal pelvis (E. truncate). In the body of animals and birds sick with eimeriosis, intoxication occurs with the products of metabolism of protozoa and putrefactive microflora, which leads to a disruption of the nervous system, causing inhibition of animals, up to a coma, muscle tremor and paralysis of the limbs. Animals lose weight; meat of ill animals has low nutritional value and quickly deteriorates during storage. In birds, egg production decreases. Farms incur large expenses for the purchase of drugs for the treatment, prevention and disinfestation of premises. With a strong degree of invasion, eimeriosis can cause mass death of animals (Akbaev M.Sh., Voseleevich F.I., Akbaev PM et al. Parasitology and invasive diseases of animals. - M .: Kolos. - 2008. - 756 p.) [1 ].

For the treatment of eimeriosis in animals and birds, a number of drugs have been proposed that have anti-eimeriotic activity: chemocid, trichopolum, salinomycin, baykoks, nitrofuran (furacrilin, furazolan, furazolidone) and sulfanilamide drugs (sulfadimezin, sulfadimethoxine, sulfaperedosoline, levine sulfin, and norvines, norin monomiecin and many others (Akbaev M.Sh., Vodyanov A.A., Kosminkov N.E. et al. Parasitology and invasive diseases of animals. - M .: Kolos. - 2000. 559 p. [2]; Pleshakov S. A., Larionov S.V., Rozovenko M.V. Efficiency of trucks krillina and furazonal in case of rabbit eimeriosis // J. “Veterinary medicine.” - No. 8. - Saratov, 1997. P.28-30 [3]; Pleshakov SA Scientific basis for the use of complex preparations based on nitrofurans in rabbit eimeriosis // Abstract of dissertation of the candidate of veterinary sciences. - 1999. - 18 p. [4]). However, no specific drugs specifically acting on eimeria have been found, and those that are widely used have one or several negative properties: high cost, toxicity, cumulation in edible animal tissues, immunosuppressive effect, the risk of developing resistant strains of the pathogen.

Despite the widespread use of drugs as a means of treatment and prevention of eimeriosis, it is not possible to obtain 100% indicators of the extent and intensity of effectiveness. Therefore, these drugs are used repeatedly. For example, to treat eimeriosis in rabbits, furazolidone is used at 250 mg / kg for 10 days, and after a 5-day break, sulfadimethoxine is used on the first day, 2 g, and in the next 9 days, 1 g per animal (Ponomarenko V.Ya. K the issue of chemoprophylaxis of rabbit coccidiosis // Materials of the scientific conference of parasitologists of the UAASSR. - Part 2. - Kiev. - 1975. - P.116) [5].

A method of treatment with furacryline or furazonal in combination with vitamins and salts of hydroxyethylidene diphosphoric acid is proposed, which allows to reduce single doses of drugs (active principle) to 30 mg / kg and 15 mg / kg of body weight, respectively (RF patent 2154470, publ. 20.08.2000) [ 6].

There is a method of treating cows with eimeriosis, which includes feeding coccidin with food and additionally administering once a 10% solution of Heraconda, a phytopreparation that stimulates metabolic processes and increases the body's resistance, at a dose of 4.5-5.5 mg / kg body weight (RF patent 2234932 , published on August 27, 2004) [7].

There is a method of treating eimeriosis in animals and birds, including twice feeding with an interval of 5 days at a dose of 30 mg / kg of xymedon or xymedone hydrochloride for small animals and additionally administering furazolidone at a dose of 20 mg / kg of body weight in a five-day course with an interval of 3 days (RF patent 2322235, published April 20, 2008, bull. No. 11) [8].

Thus, the principle of using anti-eimeriotic drugs is based on their repeated use, which is non-indifferent for any organism. Many of them have toxic, irritating and immunosuppressive effects. In addition, the prolonged use of one coccidiostatic agent leads to the appearance of stable races of eimeria, which cause the phenomena of dysbiosis, contribute to the inhibition of the enzymatic activity of the gastrointestinal tract, and can cause other functional changes in the animal organism.

In addition, when using sulfonamides, allergic reactions and other side effects can occur: dermatitis, leukopenia, neuritis. Due to the poor solubility of sulfonamides and especially their acetylation products formed in the body by replacing the hydrogen of the amino group with an acetic acid residue, they can precipitate in the kidneys in the form of crystals and clog the urinary tract (Mashkovsky M.D. Medicines. Part 2. M., Medicine, 1993, p. 688) [9].

Therefore, the search for new drugs and their application for the prevention and treatment of eimerioses of animals, birds and humans, affecting various parts of the pathological process, is an urgent task of both pharmacy and veterinary medicine.

The objective of the claimed technical solution is to create an effective non-toxic anti-emeriotic drug of a new generation - penetrating through the membrane of a parasite, which therefore has a characteristic set of properties:

- ultra low therapeutic doses;

- high LD _{50 50} 2500 mg / kg;

- high efficiency against eymeria, providing an expansion of the arsenal of known ineffective, toxic and expensive drugs for this purpose.

The technical result of the claimed invention is the creation of a new “Deprot-14” pharmaceutical composition, new in terms of the mechanism of action of non-toxic (LD ₅₀ 2500 mg / kg), effective for the treatment of eimeriosis with 2-fold administration with an interval of 24 hours at a dose of 4 mg / kg (in terms of on the active substance - phosphonium salt A). The drug is taken 1 time per day in the morning feeding for 2 consecutive days.

The claimed technical result is achieved by using the composition "Deprot-14" containing n-tetradecyl tributylphosphonium chloride (A) and 5,7-bis- (m-nitroanilino) -4,6-dinitrobenzofuroxan (B) as active components in a weight ratio of 1: 10 and glucose (excipient), as shown in Figure 1:

As we have shown, the Quaternary salts of phosphonium have high antibacterial properties due to the interaction with the lipid components of cell membranes [Galkina IV, Melnikova NB, Tudriy EV, Galkin VI, Zhiltsova O.E., Zhukova O.V., Egorova S.N. The interaction of salts of phosphonium with lipid components of the membranes / Pharmacy, 2009. - No. 4. - S.35-38]. Derivatives of benzofuroxanes have a wide range of biological activity, for example acaricidal and bactericidal [US Pat. RF 2255935, publ. 07/10/2005], fungicidal [US Pat. RF 2032678, publ. 04/10/1995]; their use is also proposed for the treatment of angina pectoris [RF application 2000132204, publ. 11/10/2002; RF application 2000132206, publ. 10/20/2002]. From the studied prior art there is no information about the presence of anti-emeriic activity in benzofuroxanes and in quaternary phosphonium salts, both individually and in combination, which is evidence of the conformity of the claimed technical solution to the “novelty” criterion for inventions. Moreover, the antibacterial, antifungal and antimicrobial properties of the above drugs are widely known [Galkina I.V., Tudriy E.V., Bakhtiyarova Yu.V., Egorova S.N., Galkin V.I. Synthesis, structure and study of the biological activity of phosphonium salts / In collection: Pharmacology and toxicology of organophosphorus compounds and other biologically active substances. - Kazan, 2008. - P.23; pat. JP 2002308713, publ. 10/23/2002; pat. JP 2005060332, publ. 03/10/2005; pat. JP 2000316701, publ. 11/21/2003]. The active components of the claimed composition based on the salt of the Quaternary phosphonium and substituted dinitrobenzofuroxan are used in the claimed technical solution for a new purpose - as an anti-eimeriotic drug, which is not obvious to a specialist in the analyzed field of technology; Thus, the claimed technical solution meets the criterion of "inventive step" for inventions.

The claimed technical solution meets the criterion of "industrial applicability" to the invention, because The claimed composition can be obtained by using known components using standard equipment and known techniques.

The essence of the claimed technical solution is to create an antiprotozoal composition containing n-tetradecyltri-n-butylphosphonium chloride and 5,7-bis- (m-nitroanilino) -4,6-dinitrobenzofuroxan as active components in a weight ratio of 1:10 and glucose as an auxiliary substance.

The claimed technical solution is as follows.

1. Get the first active component (A) of the claimed composition is n-tetradecyltriphenylphosphonium chloride of the formula:

by the method described in [Galkina I.V., Melnikova NB, Tudriy E.V., Galkin V.I., Zhiltsova O.E., Zhukova O.V., Egorova S.N. The interaction of salts of phosphonium with lipid components of the membranes / Pharmacy, 2009. - No. 4. - S.35-38]:

2. Get the second active ingredient - 5,7-bis- (m-nitroanilino) -4,6-dinitrobenzofuroxane of the formula (B), which was synthesized according to the method [L.M. Yusupova, S.Yu. Garmonov, I.M. Zakharov et al. Biological protective agents for multi-purpose use based on chlorine derivatives of nitrobenzofuroxan / Bulletin of Kazan Technological University, 2005, No. 1, pp. 103-111]:

Crystalline medical glucose was used according to GOST 975-88 as a nutrient for protozoa and filler (manufacturer Dnepropetrovsk starch-syrup and syrup factory).

The claimed technical solution is illustrated by the following examples.

Example 1. Obtaining antieimeriotic composition "Deprot-14" and dosage forms based on it

2.25 g of glucose, 0.25 g of n-tetradecyltri-n-butylphosphonium chloride A and 2.50 g of 5,7-bis- (m-nitroanilino) -4,6-dinitrobenzofuroxane B are mixed and triturated in a porcelain mortar to obtain homogeneous red powder (pharmaceutical composition "Deprot-14"). Powder is a dosage form for treating birds. For dosing in animals, cachets were filled with powder (metered closable starch containers). An 0.5 g oral dosage form contained 0.025 g of phosphonium salt (A), 0.250 g of dinitrobenzofuroxan (B), and 0.225 g of glucose.

Example 2. The study of the anti-eimeriotic activity of "Deprot-14" in relation to mixed eimeriosis of animals and birds caused by eimeria: E. stidae, E. perforans, E. magna, E. intestinalis, etc.

Testing the therapeutic effectiveness of the compound "Deprot-14" with eimeriosis of animals and birds was carried out in 3 experiments.

In the first experiment, 15 rabbits free of coccidia and helminths were used. The animals were infected with a mixed culture of sporulized Eimeria oocysts (E. stidae, E. perforans, E. magna, E. intestinalis, etc.). To do this, Aimeria non-sporulated oocysts were cultured in Petri dishes in a thermostat at a temperature of 26-27 ° C, periodically irrigated with dechlorinated water. The suspension of oocysts was washed 2 times with tap water by centrifugation and the number of oocysts in 1 ml was counted. Aimeria oocysts were calculated per each animal at a rate of 20,000 per 1 kg of body weight.

After infection, rabbits were divided into 3 groups. 10 days after infection, the animals of the first group, along with concentrated food, were given the compound “Deprot-14” at a dose of 350 mg / kg, twice with an interval of 1 day, the second group - furazolidone at a dose of 20 mg / kg, two five-day courses with an interval of 3 days. Rabbits of the third group were not treated, and they served as a control.

Animals during the experiment were in the same conditions of detention, preventing spontaneous infection. Feeding animals was carried out in accordance with zootechnical standards. A clinical examination of the livestock was performed daily. Fecal samples were taken for research before infection and drug administration and 10, 15, 30 and 45 days after treatment.

For coprological studies of material samples, combined helminthovoscopic methods were used.

Data on the anti-eimeriotic efficacy of the drugs are presented in table 1.

The table shows that before treatment, all rabbits were infected with eimeriosis, the intensity of invasion (II) ranged from 195 ± 1.1 to 218 ± 2.3.

10 days after the start of treatment in the 2 experimental groups, the AI decreased and amounted to 58.0 ± 2.5 in the first group (Deprot-14) and 92.8 ± 0.9 in the second (furazolidone). In rabbits of the control group, a slight increase in the number of oocysts was noted (228 ± 0.6). The intensivity (IE) of the Deprot-14 compound, which was used in the treatment of rabbits of the first group, was 73.6%; in the second group, IE of furazolidone was 52.4%, i.e. was lower by 21.2%. It should be noted that during this period, despite a decrease in the number of oocysts in animals of both the first and second groups, not one of the rabbits was completely freed from oocysts.

15 days after treatment in the first group, the number of oocysts was 119 ± 1.7, against 218 ± 2.3 (before treatment), IE was 45.3%. Two rabbits completely freed from oocysts; extensibility of drugs (EE) was 40.0%. In the second group, II was 135.2 ± 2.4 versus 195 ± 1.1 (before treatment). IE amounted to 30.7%, and extensibility - 20%. 30 days after treatment in the first group, the intensity of eimeriotic invasion decreased to 14.6 oocysts, the efficacy was 93.3%, and the extensivity was 60.0%. After treatment with furazolidone, the average number of oocysts in rabbits was 69.0 ± 2.5, the intensity efficiency was 64.6%, and the extensivity was 20%. After 45 days, the rabbits treated with the Deprot-14 compound were completely freed from Eimeria oocysts. In the second group (treatment with furazolidone), the invasion intensity became higher compared to the previous periods, and oocysts were isolated in all rabbits, and therefore the EE was 0%, and the intensity was 25%.

In rabbits of the control group, the intensity of eimeriotic invasion did not decrease during the experiment and amounted to 235 ± 2.1 after 15 days - 213 ± 1.4 after 30 days, 213 ± 1.9 after 45 days (against 207 ± 1.6 before treatment) .

Thus, as a result of the studies, it was found that after treatment of rabbits experimentally infected with eimeriosis, the intensity of the Deprot-14 compound was significantly higher by 21.2% after 10 days, 14.6% after 15 days, and 28 after 30 days. 7% and after 45 days - 75%. A similar pattern was revealed in determining the extensibility of drugs.

Table 1 Comparative effectiveness of the composition "Deprot-14" and furazolidone in the treatment of eimeriosis in animals and birds The number of animals in the group No. gr. II before treatment (ex) M ± m Invasion Intensity and Extensibility 10 days 15 days 30 days 45 days II (ex) M ± m IE (%) EE (%) II (ex) M ± m IE (%) EE (%) II (ex) M ± m IE (%) EE (%) II (ex) M ± m IE (%) EE (%) With eimeriosis of rabbits 5 one 218 ± 2.3 58 ± 2,5 73.6 0 119.3 ± 1.7 45.3 40,0 14.6 ± 1.8 93.3 60.0 - 100.0 100.0 5 2 195 ± 1,1 92.8 ± 0.9 52,4 0 135.2 ± 2.4 30.7 20,0 69 ± 2.5 64.6 20,0 146.2 ± 2.7 25.0 0 5 3 207 ± 1.6 228 ± 0.6 - - 235 ± 2.1 - 0 243 ± 1.4 - - 213 ± 1.9 - 0 With eimeriosis in cattle 10 one 112 ± 2.2 45.2 ± 1.9 59.6 0 26.5 ± 1.4 76.3 30,0 6.4 ± 2.0 94.3 80.0 - 100.0 100.0 10 2 98 ± 1.41 64.1 ± 0.8 4.6 0 54.3 ± 1.6 44.6 20,0 41.6 ± 1.9 57.6 30,0 52.5 ± 3.0 46,4 30,0 10 3 91 ± 1.5 115 ± 0.9 - 0 129 ± 1.7 - - 146 ± 2.3 - - 180 ± 1.4 - 0 With bird eimeriosis fifteen one 80 ± 3.1 55.9 ± 3.5 30,2 0 45.1 ± 2.6 43.6 40,0 10.9 ± 2.4 86.4 73.3 3.1 ± 2.9 96.2 86.7 fifteen 2 68 ± 3.6 58.1 ± 3.0 14.7 0 53.4 ± 1.8 21,4 13.3 43.4 ± 1.9 36,2 26.7 23.1 ± 2.2 42.7 20,0 fifteen 3 60 ± 2.9 74.3 ± 2.8 - 0 85.3 ± 3.6 - 0 92.6 ± 3.2 - 0 100.3 ± 3.6 - 0

The second experiment was carried out on calves 1-1.5 months of age, which were naturally invaded by eimeria of the species E. bovis, E. zuernii, E. ellipsoidalis, etc.

After carrying out coprological studies, animals based on analogues (having similar clinical signs, the same live weight and degree of invasion) were divided into 3 groups (10 animals each), of which two experimental and one control.

Group 1 calves, along with concentrates, were given the “Deprot-14” compound at a dose of 100 mg / kg twice with an interval of 1 day. Animals of the second group received furazolidone (20 mg / kg) in two five-day courses with an interval of 3 days. The calves of the third group served as control. Animals of all groups were kept under identical conditions, and their feeding was carried out in accordance with zootechnical standards. A clinical examination of the livestock was performed daily. Fecal samples were taken for research before drug administration and 10, 15, 30, and 45 days after treatment.

The results of the study of the anti-eimeriotic efficacy of the studied preparations in cattle are also presented in Table 1. It can be seen from it that the intensity of eimeriotic invasion in naturally infected calves ranged from 91.0 ± 1.5 to 112 ± 2.9. 10 days after treatment in animals of the experimental groups there was a decrease in the number of oocysts. However, the intensity of the drugs was different, and in the first group ("Deprot-14") it was 59.6%, in the second (furazolidone) - 34.6%.

15 days after treatment, there was a further decrease in the number of Eimeria oocysts in the animals of the experimental groups, and in the first group their number was 26.5 ± 1.4, in the second - 54.3 ± 1.6, and the efficiency was 76.3% and 44.6%, respectively. During this period, three animals from the first and two from the second group were completely freed from the eimeria.

30 days after treatment with Deprot-14, the intensity of eimeriotic invasion in the first group decreased to 6.4 ± 2.0 oocysts, the intensity was 94.3%, and the extensivity was 80%. After treatment with furazolidone, the average number of oocysts in calves was 41.6, the intensity efficiency was 57.6%, and the extensivity was 30%. After 45 days, the calves treated with the Deprot-14 compound were completely freed from Eimeria oocysts. In the second group, the invasion intensity was 52.5 ± 3.0, the intensity was 46.4%, and the extensibility was 30%. The intensity of eimeriotic invasion in calves of the untreated group over the 45 days of the experiment increased to 180 ± 1.4 oocysts, against 91.0 ± 1.5 at the beginning of the experiment.

The third experiment was carried out on 45 chickens of 25 days of age, free from parasites. In the vivarium of the Department of Parasitology, Kazan State Academy of Veterinary Medicine, the bird was infected with a mixed culture of sporulated Aimeria oocysts (E. tenella, E. acervulina, E. maxima, etc.). Material for infection was obtained from birds naturally infected with eimeriosis. Oocysts were pre-sporulated for 5 days. The bird was infected with a suspension of oocysts in an amount of 1 thousand oocysts per 1 kg of weight, after which they were divided into 3 groups (15 birds each). 6 days after infection, in the first group, the chickens received the “Deprot-14” preparation at a dose of 350 mg / kg, twice with an interval of 1 day, in the first group, in the second group, furazolidone at a dose of 20 mg / kg in two five-day courses with an interval of 3 days. A third group of chickens served as a control. The feeding and keeping conditions for chickens of all groups were the same and corresponded to zootechnical standards. Coprological studies were performed 10, 15, 30 and 45 days after the start of treatment.

The research results are shown in table 1. The data in the table show that in chickens experimentally infected with eimeriosis, the number of oocysts ranged from 60.0 ± 2.9 to 80.0 ± 3.1 before treatment. 10 days after treatment, the intensity of invasion in the first group was 55.9 ± 3.5, in the second - 58.1 ± 3.0, in the control group 74.3 ± 2.8 (versus 60.0 ± 2.9 in beginning of the experiment). During this period, complete release of chickens from oocysts did not occur. The intensity of the pharmaceutical composition “Deprot-14” was 30.2%, against 14.7 when using furazolidone. Fifteen days after treatment, a further decrease in eimeria oocysts occurred in animals of the experimental groups, and in the first group their number was 45.1 ± 2.6, in the second - 53.4 ± 1.8, and the intensity efficiency - 43.6% and 21 , 4%, respectively. During this period, some animals were completely freed from Aimeria oocysts, and the EE of the drug in the first group was 40.0%, in the second 13.3%. 30 days after treatment, the intensity of eimeriotic invasion in the first group decreased to 10.9 ± 2.4, in the second to 43.4 ± 1.9. The intensity of the drug in the first group was 86.4%, in the second it was lower by 50.2% and amounted to 36.2%. After 45 days, 86.7% of the chickens treated with the Deprot-14 preparation were freed from oocysts, and the drug's intensity was 96.2%. In the second group, the intens- and extensibility of the drug was significantly lower and amounted to 42.7% and 20.0%, respectively.

In chickens of the control group, the intensity of eimeriotic invasion during the experiment period (45 days) increased from 60 ± 2.9 oocysts at the beginning and to 100.3 ± 3.6 at the end of the experiment.

Thus, the claimed method of treatment of eimeriosis in animals and birds, which consists in the use of the pharmaceutical composition "Deprot-14", allows to achieve 96.2% -100% of the effectiveness of treatment of animals with eimeriosis.

Example 3. Determination of acute toxicity of the pharmaceutical composition "Deprot-14"

The level of acute toxicity of the claimed anti-emeriotic composition was determined in mice under vivarium conditions of the Department of Parasitology of the Kazan State Academy of Veterinary Medicine by standard methods. It was found that with intragastric administration to mice, the LD ₅₀ was 11500 ± 1281.7 mg / kg. The variability of lethal doses was determined by the ratio of LD ₈₄ (16175 mg / kg) and LD ₁₆ (5306 mg / kg). According to GOST 12.1.007-76 "Degelm-14" refers to substances of the fourth hazard class according to Izmerov [Izmerov N.F., Sanotsky IV, Sidorov K.K. Toxicometry parameters of industrial poisons with a single injection (Reference). - M .: Medicine, 1977. - S.196-197].

As a result of the study, it was found that the claimed pharmaceutical composition "Deprost-14" has high anti-eimeriotic effectiveness at low course therapeutic doses of the active ingredients of the composition (0.005 g / kg) (A) and 0.05 g / kg (B), while as sulfa drugs, for example sulfadimethoxin, they are used in doses per treatment course of 0.6 g / kg, which is 120 times the dose of the drug we offer in component (A) and 12 times in component (B). When using the composition "Deprost-14" there are no disorders of the stool and other signs of intestinal infection.

Thus, the pharmaceutical composition, including three components - n-tetradecyltri-n-butylphosphonium chloride, 5,7-bis- (m-nitroanilino) -4,6-dinitrobenzofuroxan in a certain weight ratio and glucose, is highly effective in the treatment of infected eimerioses animals and birds in low therapeutic doses.

Claims (1)

Antimeriotic pharmaceutical composition containing n-tetradecyltri-n-butylphosphonium chloride and 5,7-bis- (m-nitroanilino) -4,6-dinitrobenzofuroxan as active components in a weight ratio of 1:10 and glucose as an auxiliary substance.

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