1. Introduction

Conservative treatment of patients with single-chamber and multi-chamber forms of echinococcosis, which are among the most severe and common human helminthiasis, is still among the complex problems. The use of antiparasitic drugs albendazole, mebenda, flubendazole requires long-term treatment [1,2]. The above drugs, with daily and prolonged oral use (up to 2-3 months), cause a 90-96% delay in the growth of larvocysts, the death of individual conglomerates of the parasite [1].

The half-life of albendazole has a significant variation (from 1h to 9h) [2]. The factors determining the effectiveness of albendazole therapy, in addition to the volume, localization, multiplicity of lesions, duration of the disease and others, should include a controlled factor - the level of the drug in plasma, depending on its absorption in the intestine. In order to increase the bioavailability of albendazole, many authors suggest increasing the daily dose of the drug in the treatment of experimental echinococcosis.

A.M. Lurie (1987) proposed combining albendazole with phospholipids, which led to an increase in the bioavailability of the drug due to increased absorption from the gastrointestinal tract. At the same time, prolonged higher levels of the drug are provided in the blood. However, the search for more effective methods of treatment with prolonged-acting drugs and with fewer side effects remains an urgent task.

Currently, it has been shown that drugs enclosed in liposomes can be protected from peripheral metabolism. At the same time, their effectiveness increases due to the ability to reduce their therapeutic doses [4,5,6,7]. In this regard, in this study, the goal was to develop a liposomal form of albendazole (LFA) to increase the efficiency of transporting the drug to the area affected by echinococcus liver, increase its concentration in the pathological focus, and reduce the side effects of the drug. In this work, a comparative study of the effect of free albendazole and albendazole encapsulated in small single-layer liposomes (SSLL) on the course and development of experimental liver echinococcosis was carried out.

2. Material and Methods

Experiments were carried out on 40 Chinchilla rabbits with an initial weight of 2200-3500 gr. Modeling of experimental echinococcosis of the liver was carried out according to the method we proposed (patent for invention No. 3441- 1996). The animals were divided into 2 groups (20 in each). In group 1, animals with experimental liver echinococcosis were treated with albendazole administered orally at a dose of 250 mg/kg of animal weight for 1 month.

In group II, the treatment of animals with experimental liver echinococcosis was performed with a liposomal form of albendazole. Taking into account the fact that intraperitoneal administration of the above form of the drug reduces their capture by Kupfer cells of the liver and macrophages of the spleen by 8 times, we used an intravenous route of administration of the drug. The animals were intravenously injected with a liposomal form of albendazole at a dose of 250 mg / kg of animal weight for 1 month.

The method of preparation of the liposomal form of albendazole.

Sheep liver was used to obtain liposomes. Lipid extraction was carried out by a modified Folch method (1957). Phospholipid fractionation was carried out in thin layers of KSK grade silica gel containing 6.5% gypsum in a system of chloroform-methanol-acetic acid-water solvents in the ratio (65:43:14). To obtain small single-layer vesicles (SSLV), the lipid dissolved in an organic solvent was dried with a stream of helium gas until a thin film formed at the bottom of the tube. Albendazole, dissolved in distilled water, was added to the dried lipid. Lipid was hydrated by shaking. Thus, the prepared suspension was voiced using the UZDN-1 apparatus at a frequency of 22 kHz (with a power of 3 watts) for 5 minutes with an interval of 2-3 minutes. To obtain a homogeneous fraction of the SSLV, the liposome preparation was passed through millipore filters. A cylinder of a single syringe with a size of 5 cubic meters was used as a micro column. The column was filled with sefadex Gm10 and balanced with an appropriate buffer. A sample of liposomes was applied to the finished column. The column was placed in a suitable centrifuge rotor and rotated for 5 minutes at 200 rpm. The drug not included in the liposome was contained in sefadex, and the sample consisted of liposomes containing only the included substance, which was measured on a gamma meter. At the same time, the percentage of inclusion of the drug in the liposome was 45% of the administered dose.

The objects of the study were echinococcal cysts, their fibrous capsule, liver tissue taken at different distances from the pathological focus. The material was taken for 3, 7, 14, 21, 28 days of experience. Intact rabbits served as control. Histological methods of research were used.

3. Results and Discussion

Comparative analysis of morphological changes in the liver of animals of group I and II revealed certain differences. In group I of experimental animals, against the background of albendazole treatment, pronounced demarcation inflammation was observed around the implant, foci of hyaline-drip dystrophy of hepatocytes with necrosis appear in the depth of the hepatic parenchyma. These data indicate the toxic effect of parasitic foci of the liver.

On the 14th day in this series of animals, there is no positive clinical dynamics from the treatment. The echinococcal cyst of the liver is not reduced in size, its content is homogeneous, the chitinous membrane is thickened. There are areas of contamination of parasitic cysts throughout the parietal peritoneum, large omentum, loops of the small and large intestines. On the 21st-28th day and in later periods of treatment, a decrease in the size of parasitic liver cysts was observed, partial death of parasite elements occurred. With the parasitological express method of studying echinococcal fluid in order to detect germ elements of the parasite, protoscolexes were detected in the early stages of the experiment, and on the 21st-28th day the latter were absent. Histological examination of the liver tissue in the circumference of the parasitic cyst determines the foci of necrosis, inflammatory infiltration with a predominance of neutrophilic leukocytes. Balloon dystrophy is pronounced in hepatocytes, the nuclei are hyperchromic, sometimes in a state of karyolysis.

In the second group of animals, on the 3rd day of treatment with the liposomal form of albendazole, the absence of free fluid in the abdominal cavity was noted. In the area of implantation and inoculation, the formation of a delicate fibrous capsule around an echinococcal cyst was determined. The contents of the cysts are amber-transparent, the chitinous shell is somewhat thickened in size. No areas of contamination in the abdominal cavity have been identified. During histological examination, a demarcation shaft consisting of macrophages, leukocytes and lymphocytes is determined in the circumference of the parasitic cyst. Around the focus in the hepatic tissue, minimal changes are noted with vascular hyperemia, slight swelling of the interstitial tissue and activation of Kupfer cells. Hepatocytes retain a girder structure, their cytoplasm is in a state of turbid swelling.

On the 7th day of the experiment, the animals retain their original weight, are active, mobile, they take food well, the rabbit fur is not fallen. Upon autopsy, there is no free fluid in the abdominal cavity, the peritoneum is smooth, shiny. The parasitic cyst of the liver is reduced in size, tightly wrapped in a whitish fibrous capsule. Upon opening of the cyst, the echinococcal fluid is reduced, there is a sharp wrinkling of the chitinous membrane. Microscopy of echinococcal fluid-scolexes and protoscolexes are not detected. Liver tissue on the section of the usual color. Microscopic examination shows a thickening of the chitinous membrane of a parasitic cyst. Directly around the chitin, foci of necrosis and hemorrhage are detected, pronounced infiltration of tissue by lympho-histiocytic elements. The hepatic tissue adjacent to the described demarcation shaft is characterized by pronounced serous edema of the hepatic parenchyma and hypertrophy of Kupfer cells with an increase in nuclei in volume and hyperchromia. Balloon dystrophy is noted in hepatocytes, the hepatic beams are deformed, the cytoplasm of hepatocytes is fine-grained, there are binucleated cells.

On the 14th and 21st days of the experiment, the condition of the animals is satisfactory, they are active, gain weight up to 200-300 grams, willingly take food. The surgical wound heals by primary tension. During a macroscopic examination of the abdominal organs, it is noted that the organs have no pathological changes visible to the eye. There is no pathological fluid in the abdominal cavity, the peritoneum is smooth, shiny. The liver and spleen are not enlarged in size. The echinococcal cyst of the liver is sharply reduced in size, compacted, and also surrounded by a dense fibrous capsule. Upon opening of the cyst, the chitinous shell is sharply wrinkled and soldered to the wall of the fibrous capsule, filling of the cyst cavity with connective tissue is noted. Under microscopy, the embryonic elements of the echinococcal parasite are not detected. In most cases, on the 21st day of the experiment, complete resorption or calcification of the parasitic cyst was observed. There are no foci of insemination in the abdominal cavity. Microscopic examination showed that layers of granulation and mature connective tissue with a predominance of fibrous structures are found in the implant area. In this fibrous tissue, remnants of the chitinous shell are found in the form of a homogeneous mass, as well as detritus of echinococcus. There are small fields of lymphohistiocytic infiltration in the circumference of the fibrous capsule. Hypertrophy of Kupfer cells is noted in the hepatic tissue, the hepatocyte is in a state of turbid swelling, their nuclei are hypertrophied, which indicates a compensatory adaptive reaction of liver cells. In some cases, petrified foci of daughter echinococcal vesicles are detected in the liver tissue. In the areas of death of daughter echinococcal cysts, actively phagocytized macrophages are found, the cytoplasm of which is filled with phagocytized particles.

On the 28th day of the experiment, the animals are active, mobile, there is an increase in weight up to 300-350 grams. The peritoneum is smooth, shiny, the liver cyst is sharply reduced in size, compacted, calcified in places. After opening the cyst, the tissue is cut with difficulty, the fibrous capsule is sharply thickened. The chitin shell is lysed, replaced by dense connective tissue. Under microscopy, there are detritus masses of echinococcus in the pathological focus, as well as foci of petrification. The latter are surrounded by fibrous fibrous tissue, which indicates complete resorption of echinococcus under the action of liposomes containing albendazole with replacement of connective tissue. Around this focus and in other parts of the liver, the usual structure of the liver is noted. Only small foci of lymph-histiocytic infiltration remain around the tracts. The hepatic parenchyma retains a girder and lobular structure. Hepatocytes have a normal structure, most of them are determined to be binucleated.

The results obtained indicate that the effectiveness of echinococcosis chemotherapy with albendazole is limited and requires long-term treatment, which corresponds to the data of other researchers [1]. The use of the liposomal form of albendazole has a more effective antiparasitic effect than the free form of the drug. The mechanisms of this effect are as follows: liposomes have the ability to penetrate the cells of the reticular-endothelial system, accumulate in the liver, activate its function, lead to hypertrophy and hyperplasia, and also increase the barrier function of the liver stroma. The liposomal form of albendazole is quickly transferred to the location of the parasitic cyst of the liver and has a detrimental effect on this focus, leads to destructive changes in the elements, destroys the protoscolexes and scolexes of the parasite. Lymphoid and macrophage infiltration is observed in the circumference of the echinococcal cyst. In the hepatic tissue, the girder structure is preserved, there is an expansion of the Disse space, hypertrophy of hepatocyte nuclei.

Thus, the use of the liposomal form of albendazole opens up new prospects for conservative chemotherapy of hydatidous echinococcosis of the liver up to the cure of laboratory animals from invasion.

Information about the source of support in the form of grants, equipment, and drugs. The authors did not receive financial support from manufacturers of medicines and medical equipment.

Conflicts of interest: The authors have no conflicts of interest.