BIOLOGICAL ACTIVITY OF FENBENDAZOLE BASED ON SUPRAMOLECULAR DELIVERY SYSTEM WITH DISODIUM SALT OF GLYCYRRHIZIC ACID
Due to the wide spread of animal helminthiasis, it becomes necessary to use innovative antiparasitic drugs. Fenbendazole is widely used all over the world for chemotherapy of helminthiasis, but in some cases, it is effective only in a high dose. This study, for the first time, has shown changes of physicochemical properties, pharmacokinetic parameters and an increase in the anthelmintic efficacy of mechanochemically obtained complexes of fenbendazole with disodium salt of glycyrrhizic acid for targeted delivery. Our research aimed to increase the biological activity of a solid dispersion of fenbendazole with disodium salt of glycyrrhizic acid (SDF with Na<sub>2</sub>GA), to evaluate the solubility of SDF compositions with Na<sub>2</sub>GA, pharmacokinetic parameters, and anthelmintic efficacy for laboratory models of Trichinella spiralis and Hymenolepis nana and in field tests on sheep naturally infected with gastrointestinal nematodes and moniesia. SDF with Na<sub>2</sub>GA was obtained in one-stage process in LE-101 ball mill (Hungary). The ratio of fenbendazole (Changzhou Yabong Pharmaceuticals Co., Ltd., China) and disodium salt of glycyrrhizic acid (Yuli County Jinxing Licorice Products Co., China) was 1:10. The process continued for 4 hours at 90 rpm. Pharmacokinetic parameters of fenbendazole and its metabolites in sheep were studied by high-performance liquid chromatography-tandem mass spectrometry detection. SDF with Na<sub>2</sub>GA and the substance of fenbendazole (FBZ) was administered to two groups of clinically healthy sheep (5 animals each) once per or at the dose of 2 mg/kg of active substance. Blood samples were taken from the jugular vein 0, 1, 2, 4, 6, 8, 12, 24, 33, 48, 72, and 144 hours after administration of SDF with Na<sub>2</sub>GA and the basic drug. The absorption rate constant, absorption half-life, clearance of the drug from the blood plasma, maximum drug concentration, time to reach maximum plasma drug concentration following drug administration, elimination half-life, area under the concentration-time curve and mean residence time were calculated. The efficacy of SDF with Na<sub>2</sub>GA against Hymenolepis nana and Trichinella spiralis was studied with 50 white inbred female BALB/c mice weighing 16-18 g. The eggs of H. nana were administered intragastrically with a syringe, 200 eggs per animal. On day 13 after infection, SDF with Na<sub>2</sub>GA in 1 % starch gel was administered into the stomach of mice of I, II and III groups (10 animals each) at doses of 3.0; 2.0 and 1.0 mg/kg of active substance, respectively. FBZ was the basic drug which was applied at the dose of 2.0 mg/kg (experimental group IV). The animals of the control group received the same volume of the starch gel. Trichinella spiralis was isolated by serial passages of the first stage larvae to female rats. Before infection, the mice were kept on a starvation diet for 12 hours, and then 200 larvae were injected into their stomachs using a tuberculin syringe. On day 3 after infection, the mice were divided into four experimental and one control groups (10 animals each). SDF with Na<sub>2</sub>GA in 1 % starch gel was administered into the stomachs of mice of experimental groups I, II, and III at doses of 3.0; 2.0 and 1.0 mg/kg of active substance, respectively. The FBZ substance was administered to mice of IV experimental group at the dose of 2 mg/kg. Control group of animals received 1,5 % starch gel at the same dose. The efficacy of the drugs against H. nana and T. spiralis was determined from necropsy data. The anthelmintic activity of SDF with Na<sub>2</sub>GA was also studied on young Stavropol merino sheep in field tests (LTD Agroresurs, Samara Province, Pestravsky District) in the summer 2016-2017. SDF with Na<sub>2</sub>GA was administered per or to the animals of the experimental groups (a single application per or at the doses of 3.0; 2.0 and 1.0 mg/kg of active substance vs. FBZ at the dose of 2.0 mg/kg. The control group of animals did not receive the drugs. Anthelmintic activity of drugs was determined according to the data of necropsy of the intestines of mice and the results of studies of sheep feces samples by the McMaster method before and after administration of the drugs. The data of physicochemical studies have shown an increase in solubility, a decrease in the particle size of the compositions of SDF with Na<sub>2</sub>GA, and the formation of irregularly shaped aggregates. The pharmacokinetic parameters indicated a significant increase in the rate of absorption of SDF with Na<sub>2</sub>GA and their entry into the blood, a 2,5-fold increase in the maximum concentration of fenbendazole and its metabolites in the blood, as well as a decrease in the rate of drug elimination from the body compared to the FBZ. SDF with Na<sub>2</sub>GA (3.0; 2.0 and 1.0 mg/kg) showed 100; 98,05 and 92,74 % activity against T. spiralis , 100, 98.67 and 89.04 % against H. nana , 100, 95.37 and 92.07 % against Nematodirus spp., 100, 95.42 and 90.75 % against gastrointestinal strongylates, and 96.44, 91.61 and 81.12 % against Moniezia expansa . The FBZ (2.0 mg/kg) anthelmintic activity was 3.4 times lower than that of the same dose of SDF with Na<sub>2</sub>GA upon experimental trichinellosis of mice. Its efficacy was 28.88 % against experimental hymenolepiasis of mice. The FBZ substance showed low efficacy against Nematodirus spp. (33.33 %), other gastrointestinal strongylates (39.14 %) and Moniezia spp. (17.55 %). These findings allow us to conclude that the development of drugs based on fenbendazole solid dispersion with glycyrrhizic acid disodium salt is promising, and the production technology can be scaled up.