4.4. Antitumor Activity

In the study, avarol was used in the form of a 0.5% suspension prepared with water for injection (OOO Dalhimpharm, Habarovsk, Russia) in the presence of 1% hydroxypropyl cellulose (hyprolose, Nippon Soda CO Ltd., Tokyo, Japan). In all experiments, avarol was injected daily intraperitoneally (i.p.) at a dose of 50 mg/kg—10 µL of suspension per 1 g of animal weight. The animals in the control groups received daily i.p. injections of an equivalent volume of a 1% suspension of hydroxypropyl cellulose. All injectable suspensions were prepared ex tempore.

Female mice (F₁ (CBA × C57BL6j), 2–2.5 months, 19–23 g and CBA, 2 months, 19–22 g) were used in this study. The animals were obtained from the nursery of the Scientific Centre for Biomedical Technologies of the Federal Medical and Biological Agency of Russia. Mice were kept in the vivarium of the A.F. Tsyb MRRC in T-3 cages under conditions of natural light with forced 16x/hour ventilation at a temperature of 18–20 °C and a relative humidity of 40–70%. The animals had free access to water and PK-120-1 rodent food (OOO Laboratorosnab, Russia). The work with laboratory animals was approved by the MRRC Ethical Commission (protocol number: 1-H-00025) and was performed in accordance with generally accepted norms of animal handling based on standard operating procedures adopted at the A. Tsyb MRRC and corresponding to the rules and requirements of European Convention ETS/STE No. 123 and the international GLP standard (OECD Guide 1:1998).

Two transplantable mouse tumors were used: Ehrlich carcinoma (EC) and cervical cancer (CC-5). The tumor strains were obtained from the tumor bank of the N.N. Blokhin National Medical Research Centre for Oncology of the Ministry of Health of Russia. Before tumor grafting, the hair on the lateral surface of the right thigh was removed using a ChroMini Type 1591 trimmer (Moser, Germany). The EC strain was transplanted into female F₁ (CBA × C57BL/6j) mice by the subcutaneous injection of 2.5 × 10⁶ tumor cells in 0.3 mL of medium 199 (PanEco LLC, Moscow, Russia) to the lateral surface of the right thigh. The CC-5 strain was transplanted into female CBA mice by the injection of 100 mg of tumor tissue homogenate in 0.5 mL of medium 199 into the lateral surface of the right thigh.

Two independent experiments were performed on the selected tumor models. In the first experiment, the effect of avarol on the development of solid EC was studied. For this purpose, 33 female F₁ (CBA × C57BL/6j) mice were divided into two groups, the control (17 animals) and experimental (16 animals) groups, immediately after EC grafting. Then, from the 7th to the 20th day (14 days) following the tumor transplantation, the animals in the experimental group were intraperitoneally (i.p.) injected daily with an avarol suspension at a dose of 50 mg/kg, and the animals in the control group were injected with an equivalent volume of solvent. The effect of avarol on tumor development was assessed by the growth dynamics of the tumor. Starting 7 days after transplanting EC and then every 2–3 days, the linear dimensions of the tumor nodes in all animals were measured with a caliper, and their volumes were estimated with the approximation of V=(a⋅((b+c)/2)2)×(π/6), where a, b, and c are the orthogonal diameters. We calculated the relative volumes of the tumors normalized to the tumor volume in the animal at the treatment start day, and the tumor growth inhibition index in the animals treated with avarol was calculated as: TIi,t=(V¯C,t−Vi,t)/V¯C,t×100%, where TIi,t is inhibition index in the i-th animal at observation time t, V¯C,t is the average relative tumor volume in the control group at observation time t, and Vi,t is the relative tumor volume of the i-th animal at observation time t.

The impact of cisplatin was previously investigated and established in earlier studies [31,37]. Hence, a group that received only cisplatin was not included in the research. Additionally, therapy with avarol during a two-week period did not result in negative manifestations or death in tumor-bearing animals. Therefore, an additional control group of healthy animals was not included.

The manifestation and dynamics of the avarol effect on EC were assessed and compared by intergroup statistical comparisons of the relative volumes of tumors and indicators of tumor growth inhibition in different follow-up periods. The integral antitumor effect was evaluated by the duration of growth retention, which was estimated by the time of a 10-fold increase in the EC volume on the tumor growth curves. At the end of the experiment (the day after the last injection), the animals were removed from the experiment by cervical dislocation under ether anesthesia.

In the second experiment, the effect of avarol on the development of CC-5 was studied. For this purpose, 48 female CBA mice were divided into two groups immediately after CC-5 grafting: the control and experimental groups (24 animals in each group). Five mice from each group were selected for the morphological examination of tumor nodes on the 14th day. Then, from day 7 to 18 after the tumor transplantation, the animals in the experimental group received a daily avarol suspension at the dose of 50 mg/kg, i.p., and the animals in the control group received an equivalent volume of the solvent. The effect of avarol on the development of CC-5 was evaluated using the same methods as in the EC experiment. The integral antitumor effect was evaluated by the duration of growth retardation, which was estimated by the time for a five-fold increase in the CC-5 volume on the tumor growth curves as well as by the difference in the masses of tumor nodes in the experimental groups on the 14th and 19th days of the observation. Supplementary Data S2 provides full protocols for studying the effect of avarol on the growth of SEC/CC-5 in mice, including individual indicators of the SEC/CC-5 volume at different periods after transplantation in the mice of the experimental groups, individual indicators of the SEC/CC-5 relative growth in different periods after transplantation in the mice of the experimental groups, SEC/CC-5 growth inhibition rates in avarol-treated mice in different follow-up periods, and the effect of avarol on SEC/CC-5 growth in mice (Supplementary Data S2, Tables S1–S8).

Supplementary Data S3 provides full protocols for studying the effects of avarol on body weight in mice with SEC/CC-5 with individual indicators of the body weights of the mice of the experimental groups in different periods after SEC/CC-5 transplantation, individual indicators of the relative body weights of the mice of the experimental groups in different periods after SEC/CC-5 transplantation, and the effect of avarol on the dynamics of body weight in mice after SEC/CC-5 inoculation (Supplementary Data S3, Tables S9–S14).