RU2195280C1 - Anthelmintic agent - Google Patents

Abstract

FIELD: medicine, veterinary science. SUBSTANCE: invention relates to anthelmintic agent comprising of albendazol and copper complex as an active substance that is enclosed in microcapsules making of acetylcellulose in the ratio of albendazole, copper sulfate and acetylcellulose = 1:1:0.5, respectively. Insertion of albendazole metal complex in anthelmintic agent composite allows to enhance therapeutic activity and inclusion of albendazole metal complex in microcapsule made of acetylcellulose provides the prolonged end effect that is necessary for radical chemotherapy of larval echinococcosis. Agent provides the complete inhibition of growth of exhinococcus larvocysts and death of all population of parasite. EFFECT: enhanced effectiveness of agent. 1 cl, 3 tbl, 3 ex

Description

 The invention relates to medicine and veterinary medicine, specifically to medical and veterinary helminthology, and can be used to treat larval echinococcosis and other cestodoses, as well as human intestinal nematodoses and farm animals.

 Larval echinococcoses are severe parasitic diseases of humans and farm animals caused by the echinococcus larva and leading to the death of patients. Alveolar echinococcosis (alveococcosis) is a human disease, and hydatidosis echinococcosis is a disease of humans and farm animals. The only radical method of treating echinococcosis to date is surgical, ineffective in advanced cases of the disease. A radical operation can be performed in 11-15% of patients with alveococcosis and in 25-58% of patients with hydatidosis echinococcosis; the remaining patients in 93% of cases are doomed to die within 10 years after surgery. But even with a successful operation, cases (3-54%) of recurrence of the disease are not uncommon. Relapses lead to the need for repeated operations performed up to 10 times (B.V. Petrovsky et al., 1985).

 The first drug for the conservative treatment of larval echinococcosis was identified in experimental models of diseases in 1974 (A. I. Krotov et al., 1974). Since then, carbamatebenzimidazole preparations — albendazole (methyl-5- [propylthio] -2-benzimidazole-carbamate) and mebendazole (5-benzoyl-2-benzimidazole-carbamic acid methyl ester), which have been used for chemotherapy of larval echinococcosis, are capable of inhibiting all or all of the parasites. most echinococcus larvocysts in experimental and clinical conditions, which although it can significantly extend the patient’s life, does not guarantee the cure of the patient throughout his life. The most difficult chemotherapy for human alveococcosis and experimentally infected laboratory animals. The most resistant to chemotherapy was experimental alveococcosis of cotton rats, characterized by rapid growth of parasitic larvocysts and rapid death of animals from invasion (A.I. Krotov et al., 1974; P. Schantz et al., 1982).

 As a prototype, the work of D. Taylor et al. (1988), who attempted to treat experimental alveococcosis of cotton rats with the currently most effective anti-echinococcal albendazole. Cotton rats with an initial duration of experimental invasion of 1 month. after intraperitoneal infection with germinal elements of alveococcus, a suspension of albendazole in physiological saline was injected into the stomach in a daily dose of the active substance (DV) of 50 mg / kg for 1-6-month courses (5 days of treatment with a 2-day break for each week). The total course dose of DV was 1.0-6.0 g / kg. After the end of treatment, the autopsy of the treated and untreated control animals revealed that albendazole caused only reversible inhibition of the growth of alveococcus larvocysts in the treated animals by 80.6-96.1% compared with the control, but not the death of the parasite.

The disadvantages of the prototype are:
1) low therapeutic activity of the drug (80.6-96.1% inhibition of parasite growth, lack of larvicidal effect);
2) insufficient reliability of the criterion for assessing the effect of the drug on the growth of the parasite in the treated animals (but the intensity of the invasion in rats was determined at the time of the start of treatment; the exact term for the dissection of animals after treatment was not specified).

 The objective of the invention is to obtain a microcapsular form of the albenzazole metal complex with increased anthelmintic activity in larval alveolar and hydatid echinococcoses.

 The essence of the invention lies in the fact that, as an anthelmintic agent, a complex of albendazole-substance (AC) and copper is used, which is additionally included in microcapsules of cellulose acetate at a ratio of AC, copper sulfate (MK) and cellulose acetate as the starting products for the preparation of 1 : 1: 0.5, respectively. The introduction of the metal complex albendazole (MCA) as an anthelmintic agent as a DW increases its therapeutic activity compared to current AS, and the inclusion of MCA in microcapsules from cellulose acetate provides a prolonged target effect necessary for radical chemotherapy of larval echinococcosis.

 The proposed anthelmintic agent is prepared as follows. 0.5 parts of cellulose acetate is dissolved in 30 parts of acetone with heating and constant stirring. To the resulting solution was added 1 part of AC. The mixture was stirred under heating for 1.5 hours. 1 part of MK dissolved in water is added to the reaction product. The resulting mixture was stirred under heating for 1 hour. Then, 150 parts of liquid paraffin are added to the mixture, with which it is mixed for 30 minutes without heating. Acetone is distilled off from the resulting mixture under heating, under a shallow vacuum and with constant stirring. The resulting microcapsules are placed on a paper or glass filter and liquid paraffin is filtered under vacuum. The microcapsules remaining on the filter are washed with 4-5 portions of 30 parts in each hexane or petroleum ether. The obtained microcapsules are dried by heating for 4-5 hours. The finished product consists of granules of spherical or oval shape with a diameter of 100-200 microns of greenish color, odorless and tasteless. The output of microcapsules is 90-95%. The content of organic matter calculated from the bound AS is about 40%. The finished product is used as an anthelmintic agent orally in a mixture with vegetable oil.

 Unlike AS, which can only temporarily suppress the development and growth of Echinococcus larvocysts and does not have a larvicidal effect, microencapsulated MCA (MCMCA) not only completely and stably inhibits the growth of Echinococcus larvocysts in parasite hosts, but also causes the death of the entire echinococcus population in treated animals, providing radical cure of their invasion.

 The receipt and effectiveness of the proposed anthelmintic funds are illustrated by the following examples.

Example 1. MKMKA was prepared as follows. 0.5 g of cellulose acetate was dissolved in 30 ml of acetone in a three-neck reaction flask with a capacity of 0.5 l at 40-50 ^o C and constant stirring on a magnetic stirrer at a speed of 300-350 rpm After 30 minutes, 1 g of AS was placed in the flask and the mixture was stirred at 40-45 ^° C for 1.5 hours. Separately, in a 50 ml beaker, 1 g MK was dissolved in 5 ml of water at 40 ^° C. After the MK solution was cooled to 18-20 ^° C, it was placed in a 25 ml separatory funnel. Then the MK solution was added dropwise to the reaction flask, and the resulting mixture was stirred for 1 hour at 40-50 ^° C. At the end of the exposure, 150 ml of liquid paraffin were added to the flask. The mixture was stirred for 30 minutes without heating. A water-jet pump was connected to the flask, and when heated to 40-50 ^° C, under shallow vacuum and constant stirring, acetone was distilled off. The resulting microcapsules were discharged from the flask onto a ceramic with filter paper or a glass filter 1-2 and liquid paraffin was filtered under vacuum. The microcapsules remaining on the filter were washed with 4-5 portions of hexane or petroleum ether, 30 ml in each portion. Vaseline oil and hexane wash fractions after distillation were collected for reuse. The obtained microcapsules were dried at 50 ^o C for 4-5 hours. Dried microcapsules were used as an anthelmintic in a mixture with vegetable oil.

 The therapeutic activity of MCMCA was studied in experimental larval alveococcosis of cotton rats. AS and MCA (prepared as described above without the addition of cellulose acetate) were used as comparison preparations. Under the same experimental conditions, the effectiveness of the two MKMKA samples obtained by the above method was also evaluated, upon receipt of which the ratio of the starting products AS: MK: cellulose acetate was 1: 1: 0.4 and 1: 1: 0.6, respectively.

 In the experiment, cotton male rats infected at the age of 1-1.5 months were used. intraperitoneally with protoscolexes and microscopic acephalocysts of the alveococcus of the Kamchatka isolate (500 protoscolexes and 100 acephalocysts per animal) from an experimentally infected cotton donor rat. 23 days after infection, the animals were divided into 8 groups of 7-9 animals each. Rats of the first group were opened to determine the initial intensity of invasion (at the time of treatment). The average weight of the developed in their abdominal cavity larvocyst alveococcus (LA) by this time was 2.44 g per 1 rat. Rats of 6 experimental groups were treated with MKMKA and comparison drugs. In the control group were infected untreated animals.

The preparations were administered to animals mixed with refined sunflower oil or 1% starch gel in the stomach using a syringe and a metal cannula 1 time per day in gradually increasing daily doses of DV from 0.05 to 0.30 g / kg for 42 days without breaks. The regimen for changing the daily doses of DV during the course of treatment was as follows: I week - 0.05 g / kg, II week - 0.10 g / kg, III and IV weeks - 0.20 g / kg, V and VI weeks - 0 30 g / kg All animals of the experimental and control groups were opened 74 days after infection and the weight and viability of the detected LAs were determined in each. The therapeutic activity of the tested drugs was determined by the index of chemotherapeutic activity index (IHTA) and the data of a microscopic examination of native LA drugs for the presence and severity of destructive changes in the germinal elements of the parasite (protoscolexes and acephalocysts). IHTA was calculated as a percentage according to the formula: IHTA = (Mk-Ml) / (Mk-Mi) 100,
where Mi, Mk and Ml are the average values of the mass of LA per 1 animal of the original, in control and treated rats, respectively.

 The results of the experiment showed (Table 1) that the MKMKA sample was most effective, upon receipt of which the AC: MK: cellulose acetate ratio was 1: 1: 0.5, respectively (ICTA = 100.3%; pronounced destruction of the germinal elements in all aircraft all treated animals). MKMKA samples obtained at a ratio of AS: MK: cellulose acetate 1: 1: 0.4 and 1: 1: 0.6 were less effective (ICTA = 95.9 and 93.1%, respectively) and did not cause the death of all germ elements in LA in treated rats. Next in the series of decreasing efficiency were MCA (IHTA = 84.3%), AS (IHTA = 72.0%), tested under the same conditions in a mixture with vegetable oil. The lowest therapeutic activity was observed in AS administered in starch gel (IHTA = 48.4%). Moreover, in rats treated with AS with vegetable oil and starch gel, the majority of germ elements in LA remained viable.

 Example 2. MKMKA was obtained in the same way as in example 1 with a ratio of AC: MK: cellulose acetate equal to 1: 1: 0.5, respectively. The therapeutic activity of MCMCA was studied in case of larval alveococcosis of white mice at a late stage of invasion.

 In the experiment, outbred male mice infected at the age of 1.5 months were used. intraperitoneally by protoscolexes and acephalocysts of the Kazakhstan isolate (1000 protoscolexes and 200 acephalocysts per 1 animal). 121 days after infection, 5 mice were opened to determine the initial intensity of invasion. By this time, the MI indicator was equal to 7.65 g per 1 mouse. 8 mice of the experimental group were injected with MCMCA mixed with refined sunflower oil in the stomach 1 time per day for two 10-day courses in gradually increasing daily doses of DV from 0.05 to 0.24 g / kg with an interval between courses of 15 days. The total duration of treatment was 35 days (from 122 to 157 days after infection). The total dose of DV was 2.45 g / kg. 12 infected untreated mice served as control. Mice of the experimental group were opened 198 days after infection (42 days after treatment). The mice of the control group were opened as they naturally died from alveococcosis, and the surviving animals on the same day as the mice of the experimental group. The therapeutic activity of MCMCA was evaluated in the same manner as in Example 1.

 The experimental results showed (Table 2) that MKMKA showed high therapeutic activity, as evidenced by complete inhibition of growth and collapse of LA (ICTA = 183.5%), as well as the death of all LA. At autopsy in treated mice, LA conglomerates were represented by amorphous yellow structureless formations that fragmented when trying to remove them from the abdominal cavity of animals. Microscopy of the contents of these formations, represented by caseous detritus, revealed a lot of deformed or destroyed protoscolexes and acephalocysts with far-reaching destructive changes. Microscopy data showed that the death of LA in treated animals occurred much earlier than the term of dissection of animals. The mass of necrotic LA in the treated animals ranged from 3.04 to 7.13 g and averaged 4.16 g per mouse. Reliability of the radical therapeutic effect of MKMKA confirmed the long term opening of treated animals after treatment (about 1.5 months). Of 12 control mice, 10 animals died from alveococcosis 146-187 days after infection. LA in animals of the control group was represented mainly by mature, intensely budding forms containing many living protoscolexes and acephalocysts. The weight of LA in control mice averaged 11.83 g per 1 animal.

 Example 3. MKMKA was obtained in the same way as in example 1 with a ratio of AC: MK: cellulose acetate equal to 1: 1: 0.5, respectively. The therapeutic activity of MKMKA was studied in case of larval hydatid echinococcosis of white rats at a late stage of invasion.

 The invasion model was reproduced in young outbred rats of both sexes with a body weight of 140-180 g by operative intraperitoneal implantation of larvocysts of hydatidosis echinococcus (LHE) from outbred donor mice infected intraperitoneally with protosolexes from LGE from the operated person and opened after 15 months. after infection. 7 recipient rats labeled with an individual label were implanted with intraperitoneal mature LHEs (containing visually distinguishable brood capsules with protoscolexes on the inner surface of the laminate). The operation was performed under ether anesthesia; the anterior abdominal wall of rats was dissected in layers along the midline from the xiphoid process (incision length 3-4 cm), donor LHEs were inserted into the abdominal cavity and the abdominal wall was sutured in layers. To prevent peritonitis, animals were injected intraperitoneally once with 5 ml of physiological saline with antibiotics (2000 IU / ml of penicillin and 0.3 mg / ml of gentamicin. 4 rats of the experimental group 119 days after surgical implantation of 1-4 LGE with a diameter of 11-18 mm MKMKA was administered in a mixture with sunflower oil into the stomach 1 time per day for one 6-day course in gradually increasing daily doses of DV from 0.05 to 0.15 g / kg (total course dose of DV - 0.6 g / kg) .3 untreated rats, which were implanted in the abdominal cavity of 1-3 LHE diameter 11-21 mm, served as a control.The treatment efficacy was evaluated by ultrasound (ultrasound) of each rat of the experimental and control groups using an Acuson-512 ultrasound scanner (USA) 119 and 156 days after infection and according to macroscopic and microscopic studies of native LHE at autopsy of animals of both groups, carried out 182 days after infection.

 The results of the experiment showed (Table 3) that MCMCA caused the death and subsequent collapse of all LHEs in all treated animals. Ultrasound indices of the abdominal cavity of rats of the experimental group on the day the treatment was started and 37 days after the end of the treatment course showed a pronounced violation of the LHE structure (volume reduction, shape change, blurring of LHE contours, the appearance of dense inclusions in their cavities, detachment of the parenchymal layer from the cuticular membrane) . In one treated animal (rat 4), ultrasound did not reveal one of the 4 implanted LHEs 37 days after the end of treatment, while on the day of treatment with this method all implanted LHEs were detected (a sign of complete loss of hydatide fluid due to LHE collapse). Ultrasound of control rats revealed no destructive changes in all implanted LHEs. High informativeness of ultrasound was confirmed by the results of macroscopic and microscopic studies of LHE in treated and control rats at autopsy of animals after 6 months. after infection. In rats of the experimental group, opened after 2 months. after completion of the course of treatment, all LHEs were completely collapsed and contained dead and destroyed protoscolexes. In control animals, all LHEs were alive, had normal turgor, and contained brood capsules with live mature screwed protoscolexes (2-16 protoscolexes in each brood capsule).

 Thus, the microencapsulated metal complex of albendazole with the ratio of albendazole: vitriol: cellulose acetate as the starting products for the preparation of 1: 1: 0.5, respectively, is highly effective against larval hydatid echinococcoses that are not radically chemotherapy (complete inhibition of the growth of larvocyst echinococcus destructive effect on the entire parasite population in an invaded host) in comparison with that of the most effective anti-echinococcal prep so far rata albendazole (81-96% inhibition of the parasite growth, lack of larvicidal effect), which indicates a greater bioavailability of the claimed agent. It is known that albendazole and other drugs representing the carbamatebenzimidazole group are used in the treatment of not only echinococcosis, but also other larval cestodoses, being primarily effective in intestinal nematodoses of humans and farm animals. However, along with larval cestodoses, some intestinal nematodoses (trichocephalosis in clinical, and especially in experimental conditions) are poorly suited to chemotherapy with these drugs. In view of this, the use of the proposed anthelmintic agent will increase the effectiveness of chemotherapy for larval cestodoses and intestinal nematodoses of humans and farm animals.

Sources of information taken into account when preparing the application:
1. Taylor DH, Morris DL, Richards KS, Reffin D. Echinococcus multilocularis: in vivo results with albendazole and praziquantel. Trans. Roy. Soc. Trop. Med. and Hyg., 1988, v. 82, 4, p. 611-615 (prototype).

 2. Krotov A.I., Chernyaeva A.I., Kovalenko F.P., Bayandina D.G., Budanova I.S., Kuznetsova O.E., Voskobojnik L.V. Experimental therapy of alveococcosis. Message II. Efficiency in laboratory animal alveococcosis of some anti-nematode agents. Honey. parasitol. and parasitic. diseases, 1974, 3, p. 314-319.

 3. Petrovsky B.V., Milonov O.B., Deenichin P.G. Surgery of an echinococcosis, 1985, M., 216 p.

 4. Schantz P. M., Van den Bosche H., Eckert J. Chemotherapy for larval echinococcosis in animals and humans. Report of a workshop. Z. Parasitenkd., 1982, v. 67, p. 5-26.

Claims (1)

 An anthelmintic agent including benzimidazole carbamate, characterized in that it contains an albendazole and copper complex as an active substance, which is additionally included in microcapsules of cellulose acetate at a ratio of aldendazole, copper sulphate and cellulose acetate of 1: 1: 0.5, respectively, as its starting products receipt.

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