Toxicological evaluation of Supramil® tablets, an anhelmintic drug for dogs and cats

The article presents the results of studies of the toxicological characteristics of the drug for veterinary use Supramil® tablets based on R-praziquantel and milbemycin oxime. The results of toxicometry and observational data on experimental animals make it possible to attribute the finished dosage form of the medicinal product Supramil® tablets to hazard class IV “Substances of low hazard” in accordance with GOST 12.1.007 – 76. The intragastrical LD50 for mice is over 12000 mg/kg BW, and the LD50 for rats is over 15000 mg/kg BW. The drug Supramil® tablets at doses of 1500 mg/kg, 750 mg/kg and 300 mg/kg, when administered daily orally to white outbred rats for 14 days, did not cause significant changes in their clinical condition and morphology of internal organs, hematological and biochemical blood parameters.

The drug does not have allergenic properties. Evaluation of the ability of the drug to cause a state of hypersensitivity did not reveal any allergenic properties in conjunctival and nasal tests, as well as in the reaction of indirect mast cell degranulation. The results of this experimental study allow us to conclude that the medicinal preparation Supramil® tablets does not have toxic and allergenic properties. Key words: Supramil® tablets, R-praziquantel, milbemycin oxime, LD50, mice, rats. DOI:10.30896/0042-4846.2022.25.6.55-62

Anthelmintic drugs of both Russian and foreign manufacture are used for treatment and prevention of helminthiases in cats and dogs. However, as practice shows, some of them have certain disadvantages. Taking this into consideration, the development of new effective anthelmintic drugs for small domestic animals with enhanced safety profile remains relevant. Based on the RPC SKiFF's formulation [6], specialists of Astrapharm Research and Innovation Company have developed Supramil® tablets, a drug intended for treatment of cats and dogs with general nematodoses, cestodiases and mixed invasion, as well as for prevention of dirofilariasis. Active pharmaceutical ingredients of the drug are R-praziquantel and milbemycin oxime. Praziquantel contains R-praziquantel active levorotary isomer and S-praziquantel inactive dextrorotary isomer in almost equal proportions [11, 12, 15]. The cestocidial effect of praziquantel is delivered only by Rpraziquantel while S-isomer has almost no anthelmintic activity [13, 15 – 17, 19]. When using R-praziquantel, the reduction of its clinical dose down to 50% of common racemic praziquantel dose is justified while retaining its high effectiveness [14, 16 – 18].

Prazitel® Special in the form of a suspension has become the first R-praziquantel combined containing anhelmintic for cats and dogs in the world's veterinary practice [3–5, 7]. Milbemycin oxime is a semisynthetic derivative of natural macrocyclic lactone received from enzyme activity of Streptomyces hygroscopicus var. аureolacrimosus; it is active in relation to nematode larvae and imago which parasitize in gastro-intestinal tract of cats and dogs as well to invasion larvae (L3) of Dirofilaria spp. As the assessment of their toxicological properties is an obligatory stage in the development of new pharmaceutical forms introduced into clinical veterinary practice, the purpose of this work is to determine the parameters of acute and subchronic toxicity as well as the allergenic effect of the drug Supramil® tablets using standard experimental data.

Materials and Methods

The tests were conducted on laboratory animals in environment of Stavropol State Agrarian University, Federal State-Funded Educational Institution of Higher Education, in accordance with an Order [8], Guidelines [9, 10] and PHARMACOLOGY AND TOXICOLOGY 2 GOST [2]. 1 tablet of Supramil® test drug weighing 309 mg contains 62.5 mg of Rpraziquantel, 12.5 mg of milbemycin oxime and excipients. The tablets are film-coated with a flavor additive which makes them more attractive for animals.

Acute oral toxicity of Supramil® drug was determined on white outbred mice (n=60, weight 18 to 20 g) and white outbred rats (n=60, weight 190 to 220 g) of both genders. After 14- days quarantine and clinical examination the animals were divined in six groups: one control group and five experimental groups. All animals received identical care.

Before the experiment all animals had a 6-hour starvation diet. The tablets were prepared in the form of suspension in 1-% starch solution via ex tempore dispersion method. The tested drug was injected into the stomach of the experimental groups using metal atraumatic probe in doses 3,000, 5,000, 7,500, 10,000 and 12,000 mg/kg BW for mice and 3,000, 6,000, 9,000, 10,000 and 15,000 mg/kg BW for rats. The highest doses were given divisionally with 1 hour interval. The animals of control groups were given 1- % starch solution in the same way. The animals were followed up for 14 days. At the same time, daily in the morning and in the evening, their appearance and behavior, state of body hair and visible mucous membranes, feed intake, mobility, expression of intoxication and its severity and reversibility, terms of possible death were evaluated.

To calculate LD50 and other acute oral toxicity parameters of Supramil® tablets drug the probit analysis method according to Litchfield and Wilcoxon [1] was used.

To study subchronic oral toxicity of the tested drug, white outbred rats of both genders with body weight of 190 to 220 g were divided into four groups (three experimental and one control group), 10 animals each. After a 6-hour starvation diet, animals of experimental group were given Supramil® individually and orally daily for 14 days in the form of suspension prepared on 1% starch gel in 1/10 LD50, 1/20 LD50, 1/50 LD50 doses based on the previously determined LD50.

The animals of the control group were given the highest dose of 1-% starch solution for 14 days.

For the whole duration of the experiment (14 days) the animals have shown no signs of intoxication. On day 15, half of the rats from each group were euthanased followed by morphological and biochemical blood examination, autopsy, macroscopic description of organs and tissues, determination of mass coefficients of the organs (liver, heart, spleen, lungs, kidney) to reveal possible acute pathological processes within their bodies after repeated doses of intragastrically administered drug. On day 25 of the experiment, the second half of the animals was euthanased to determine the reversibility of these processes.

Obtained data was processed using Student-Fisher variation statistics method with ttest.

To determine allergenic properties of Supramil® tablets drug 40 guinea pigs of both genders with body weight of 250 to 300 g were selected, then they were divided in three experimental and one control groups, 10 animals each, complying with the principle of equivalents. Animals of the experimental groups received the drug orally for 30 days with 2.5 mg/kg R-praziquantel and 2.0 mg/kg milbemycin oxime doses. It was not given to control animals. The sensitization properties of this drug were tested on day 10, 15 and 30. Possible allergenic effect of Supramil® drug was tested using "conjunctival test" and "nasal test" models, as well as using the indirect mast cell degranulation test.

Conjunctival test was conducted a day after the last drug administration as follows: one drop mixed with distilled water in a ratio of 1:10 was injected under the upper eyelid of the right eye of the animal, and the same volume of distilled water in the left eye serving as a control. The reaction was observed after 15 minutes (rapid response) and after 24 hours (delayed hypersensitivity) and was appraised by points:

• slight reddening of the lacrimal duct;
• reddening of the lacrimal duct and sclera towards the cornea;
• reddening of the entire conjunctiva and sclera.

During the nasal test, one drop of the drug mixed with distilled water in a ratio of 1:10 was instilled in one nostril using a pipette. If, after 10 – 20 min following the administration, there were any signs of hyperemia of the mucous membrane, sneezing, swelling or increased secretion of mucus, the test was considered positive.

Serum from 10 sensitized and 10 control animals was used in order perform indirect mast cell degranulation test. Mast cells were extracted from peritoneal washings of 2 intact rat males with body weight of 300 g after administering of 8 to 10 ml of sterile saline solution with heparin heated to 37°C. Then 0.03 ml of the studied serum of test rats’ blood, mast cells and the tested drug in 1:100 dilution were applied to slide plates previously stained with 0.3% alcohol solution of neutral red. Blood serum and mast cells of control group animals and the tested drug in 1:100 dilution were used as a control test. Preparations were covered with a coverslip with its sides smeared with vaseline, then it was incubated in a thermostat at 37°C for 15 min. Then, using a microscope with 20X magnification, 100 cells were counted, among which the degranulated ones were determined. The reaction was considered negative if the count of degranulated mast cells did not exceed 10%.

Research findings and discussion

Oral administration of the drug in the highest possible dose and concentration did not result in death of mice and rats, therefore the LD50 is impossible to determine. Based on the data obtained, LD50 of the Supramil® tablets is over 12,000 mg/kg for mice and over 15,000 mg/kg for rats. This makes it possible to attribute the finished dosage form of the Supramil® tablets drug as falling under hazard class IV (“Substances of low hazard”) in accordance with GOST 12.1.007 – 76.

The drug Supramil® tablets, when repeatedly administered to white rats of the control group in all doses tested, does not affect their clinical status. Behavioral reactions, feed intake, drinking of water and breathing rate of the animals remained within the limits and did not differ from those of the control group.

During the observation period, the animals also did not show any digestive or urinary disorders. The rats gained weight equally regardless of the drug dose (Table 1).

When evaluating the effect of the tested drug on rats' CNS functional state it was noted that they retained motion activity comparable with control animals; their response to external stimuli was appropriate. The results of hematological analysis of rats' peripheral blood conducted after 1 and 10 days after the last administration of Supramil® have shown that the drug in tested doses range did not have significant effect on red and white blood cell count or the white blood cell differential (Table 2). Slight variations of hematological values corresponded to the reference values for this animal species.