This modern literature review covers the results of study of the tubulin inhibitors, mainly combretastatins A-4 and A-1 (CA-4 and CA-1) or colhicinoids. The article presents data of SAR (Structure Activity Relation) study of numerous CA-4 analogues as well as mechanisms of their action evaluated in vitro (using cultured tumor cells) and in vivo (using animals with transplanted murine tumors or with xenografts of human tumors of various histogenesis). The phosphate CA-4 derivative (CA-4P) is characterized as a vascular disrupting agent (VDAs). Approaches are described for developing CA-4 analogues stable in cis-configuration as well as methods for enhancing hydrophility of promising derivatives along with retention of their high cytotoxicity. The results of various clinical trials both of CA-4P and CA-1P administered individually or in combination with chemotherapeutic drugs are also presented. Our conclusion is that despite numerous studies performed during the last thirty years no ideal water-soluble molecule with stable cis-configuration and high cytotoxic activity has been obtained which could become the basis of an active anti-tumor medicine.

The aim of the review is to present the systematic data on antitumor activity of combretastatin CA-4 and CA-1 analogues as well as the modes of their modification and therapeutic usage.

Background. Plasma circulating tumor DNA (ctDNA) is a potential marker for tumor process monitoring. However, the feasibility of using mouse syngeneic subcutaneous melanoma model to assess ctDNA levels remains unclear.

Aim. To evaluate the feasibility of ctDNA detection in mouse B16-F10 melanoma model using droplet digital polymerase chain reaction (ddPCR).

Materials and methods. We developed and validated a ddPCR assay to quantify plasma ctDNA from B16-F10 cells. To form experimental tumors, C57Bl6 mice were inoculated with B16-F10 cells. On the first, 7th, 14th , 21st days after tumor inoculation blood was collected by retroorbital sinus puncture. ctDNA was extracted from blood plasma. On the 21st day after tumor inoculation mice were sacrificed.

Results. We validated a ddPCR assay to quantify plasma ctDNA in B16-F10 melanoma syngeneic model in C57Bl6 mice. The assay linear range was 0.5–32 copies/ul, R2 = 0.997. The empirical limit of detection of ctDNA was 1 copy/ul in 5 ng normal tissue genomic DNA background. The coefficient of variation values ranged from 44.5 % (1 copy/ul) to 16.6 % (16 copies/ul). Plasma ctDNA was detected on 21st day after tumor inoculation in B16-F10 melanoma syngeneic subcutaneous model (p = 0.004). ctDNA concentration positively correlated with tumor volume (ρ = 0.95, p = 0.05) and total circulating DNA concentration (ρ = 1, p = 0.0).

Conclusion. B16-F10 melanoma syngeneic subcutaneous model in C57Bl6 mice can be used to monitor cDNA in studies of new approaches for melanoma treatment.

Background. In non-clinical studies of antitumor agents, the use of various sources of tumor material and transplant sites significantly affects the growth parameters of syngeneic tumors, their invasive potential, and the profile of metastasis.

Aim. To evaluate the growth kinetics of syngeneic melanoma B16 in C57BL/6 mice after orthotopic (intradermally) and intramammary (into the mammary fat pad) transplantation of tumor material obtained by different methods.

Materials and methods. The experiment was carried out on mature C57BL/6 female mice. There were six groups with eight animals each. Groups 1–3 were transplanted with isografts of melanoma B16 (50 % cell suspension from tumor fragments), groups 4–6 were inoculated with cancer cells from primary culture, cultured in vitro to the early (P6) and late (P14) passages. Inoculation was performed intradermally or into the mammary fat pad.

Results. It was found that the method of obtaining tumor material (isograft or cell line), the number of in vitro passages and the site of transplantation (intradermally or intramammary) significantly affects the phenotypic features of melanoma B16 after inoculation with animals. As a result of clonal selection in vitro and in vivo, significant differences were observed in the time of appearance of the measured tumors, growth kinetics and metastasis profile. Intramammary tumor transplantation provided a reduction in the incidence of ulcers (tumor necrosis) compared with intradermal transplantation, regardless of the source of tumor material. At the same time, the development of tumor ulceration did not significantly affect the life span of animals.

Conclusion. The results of the study complement the existing data that B16 syngeneic melanoma cells have both hereditary and selective phenotypic characteristics that affect their ability to metastasize. Our data can be used in the routine work of preclinical centers for the purpose of planning and conducting studies using syngeneic tumor models.

Background. Inactivating mutations in Chek2 and Gprc5a genes are known to be associated with cancer development. Experimental carcinogenesis studies in genetically modified mice generate new data on their influence on pathology development.

Aim. In the present study in a model of lung carcinogenesis, survival parameters as well as tumor multiplicity and size in mice with Chek2 and Gprc5a heterozygous inactivating mutations were evaluated.

Material and methods. F2 hybrid mice from crosses between CBAB6F1 males heterozygous for the studied mutations and wild-type BALB / c females were used: Chek2^dAA-carriers (76 males and 64 females) and Gprc5a^insA-carriers (60 males and 42 females). Starting at four months of age, mice received urethane (ethyl carbamate) intraperitoneally at a dose of 600 mg / kg weekly for 6 weeks. After genotyping by allele-specific PCR, animals were allocated to groups. Carcinogenesis parameters were evaluated 40 weeks after the beginning of the experiment.

Results. The proportion of mice with mutations surviving to the age of three months roughly followed the Mendelian distribution (35 / 41 males and 33 / 31 females) for the offspring of males heterozygous for Chek2^dAA and was significantly lower in the case of Gprc5a^insA (20 / 40 males and 17 / 25 females, p = 0.043). The death of Gprc5a^insA carriers during the experiment was also higher than in the control group (p = 0.0506 in females). Synchronous lung and thymus neoplasms were found in 2 out of 4 Gprc5a^insA females that died before the end of the experiment, which were not found in other groups. At the end of the experiment, no significant differences in tumor multiplicity, mean linear size, and volume were found between the groups of mice with and without mutations.

Conclusion. It was found that heterozygous inactivating mutation Chek2^dAA does not affect early age development and does not modify the parameters of induced lung carcinogenesis in mice. Heterozygous carriage of Gprc5a^insA mutation in mice increases the risk of early death and sensitivity to the toxic and carcinogenic effects of urethane.

Background. In patients with HER2-positive breast cancer (HER2+ BC), as a result of targeted anti-HER2 therapy, the remission period is significantly prolonged. However, resistance to therapy often develops, especially in relation to brain metastases. In the search for effective additional drugs in combination therapy, it is advisable to study the antitumor effect of lomustine (CCNU) in HER2+ BC. Lomustine is not targeted, but is used to treat brain metastases, because it is known as one of the standard remedies for glioblastomas.

Aim. Evaluate the inhibitory activity of lomustine on the growth of spontaneous HER2+ BC in female FVB/N mice.

Materials and methods. Transgenic mice HER2+ aged 5–6 months were used, from which seven pairs of animals were selected (for control and treatment with lomustine) with spontaneous mammary tumors of the same size in each pair. Lomustine was administered once at a dose of 50 mg/kg (orally by gavage) followed by a 30-day follow-up. The criteria for evaluating the antitumor effect were ITG  % (inhibition of tumor growth), as well as TGI (tumor growth index), calculated by the ratio of the area (S) under the kinetic curve of tumor growth in the group of animals treated with lomustine (SE ) to control (SC ). With such a comparison in the control, TGI will always be 100 %.

Results. During the observation period, the volume of ВС in the control (solvent administration) increased 8-fold, while in mice treated with lomustine, the tumors regressed significantly. ITG after treatment with lomustine was 78–92 % (p = 0.007–0.0006, to control). The total mean S significantly differed by 5.6 times when comparing the control and lomustine groups and TGI in mice in the lomustine group was 18.8 %, while in the control it was 100 % (p = 0,0006).

Conclusion. Lomustine exhibits a pronounced inhibitory effect on the growth of spontaneous HER2+ ВС in FVB/N transgenic mice. The results indicate the advisability of using lomustine in patients with HER2+ ВС complicated by cerebral metastases.

Background. Streptococcus pneumoniae (S. pneumoniae, pneumococci) is an opportunistic bacterium that causes inflammatory diseases in humans. One of the virulence factors of S. pneumoniae is pneumolysin (Ply), a cholesterol-dependent hemolytic toxin that interacts with cholesterol in eukaryotic cell membranes, forms pores and leads to cell destruction and death.

Aim. Immunochemical characteristics of pneumolysin and evaluation of its cytotoxic effect in the culture of Chinese hamster ovarian cells CHO-K1.

Materials and methods. To obtain Ply, the S. pneumoniae serotype 3 strain was cultivated in brain heart broth, bacterial cells were pelleted by centrifugation and subjected to ultrasonic disintegration. The presence of pneumolysin in the resulting preparation was confirmed by immunoblotting with monoclonal antibodies to recombinant pneumolysin. The cytopathogenic effect of Ply was studied in a culture of proline-dependent epithelial-like Chinese hamster ovary cells CHO-K1 in RPMI-1640 medium. The hemolytic activity of Ply was assessed in a reaction with mouse erythrocytes.

Results. Using ultrasonic disintegration of cells of the S. pneumoniae serotype 3 strain followed by precipitation with ammonium sulfate, a protein that formed a band on electrophoresis at a level corresponding to the molecular weight of Ply (53 kDa) was obtained. The protein possessed the ability to lyse mouse erythrocytes. The authenticity of Ply was confirmed by immunoblotting with monoclonal antibodies to recombinant pneumolysin. Ply had a cytopathogenic effect on Chinese hamster ovary CHO-K1 cells. The minimum dose of the protein that induced a toxic effect on cells, manifested in the appearance of round and small cells along with normal cells, was 16.4 μg/ml per protein. When the concentration was increased up to 65.6 μg/ml, only small round cells were detected. Further increase in concentration resulted in complete destruction of CHO-K1 cells.

Conclusion. Pneumolysin can be used to develop new drugs for the treatment of pneumococcal infections.

Background. GML-3 has both anxiolytic and antidepressant effects. However, like about 70 % of аctive pharmaceutical ingredient (API) being developed, GML-3 is practically insoluble in water, which can negatively affect bioavailability. The creation of solid dispersions (SD) by crystallization with eutectic or the transfer of API to an amorphous state is one of the promising methods that allows to achieve high solubility and dissolution rate of API.

Aim. To study the crystallinity and stability of amorphous solid dispersions of GML-3 with polyvinylpyrrolidone (PVP), as well as the release of GML-3 from SD in the Dissolution test. The objects of the study were API GML-3, PVP and SD GML-3.

Materials and methods. SD was created by the method of “solvent removal” using ethanol (GML-3 is soluble in ethanol at a ratio of 1:7) and PVP with a molecular weight of 24–27 kDa. The crystallinity and stability of GML-3 API in SD were studied by differential scanning calorimetry and X-ray phase analysis. The level of release of API GML-3 from SD was evaluated spectrophotometrically at λ = 256 nm.

Results. In SD (1:5, 1:10), crystallization foci were formed, leading to the re-crystallization of API GML-3 during the solvent distillation process (for a concentration of 1:5) or a rapid loss of homogeneity due to the formation of zones of increased polymer content and API (for a concentration of 1:10). In ratios of 1:15.20, SD had high stability (after holding for 146.5 hours at 55 °C) and a high level of release of GML-3 API into purified water (96 %).

Conclusion. Stable amorphous TD GML-3 at API: polymer ratios = 1:15.20 provide a high level of release of GML-3 into purified water, which allows us to conclude that the use of TD GML-3 is promising in the development of dosage forms (tablets and capsules).

Background. Test Dissolution, which confirms the release of the active substance, is one of the most important criteria for the quality of tablets. This test significance is determined by potential correlation between the drug absorption in vivo and its release from the dosage form. It is especially difficult to develop the test Dissolution for substances that are poorly soluble in water.

Aim. To develop a test Dissolution method for cyphetrylin tablets, 60 mg and to study the release of active substance.

Materials and methods. In the work we used an original analogue of somatostatin – cyphetrylin, synthesized in the laboratory of chemical synthesis of N.N. Blokhin National Medical Research Center for Oncology of the Russian Ministry of Health. The test was developed for cyphetrylin tablets, 60 mg. The study was carried out on a dissolution tester Erweka series 700 (type II – Paddle (Erweka, Germany), using hydrochloric acid and isopropyl alcohol and their mixture as a dissolution medium. Quantitative determination of released cyphetrylin was carried out on a Agilent Cary® 100 recording spectrophotometer (Agilent Technologies, USA).

Results. Optimal conditions were selected to determine the release of cyphetrylin from tablets, 60 mg. Since cyphetrylin is insoluble in water, it is proposed to use a mixture of hydrochloric acid and isopropyl alcohol in a ratio 3:2 as a dissolution medium. It was shown that a paddle rotation speed of 100 rpm provides the release of cyphetrylin normalized quantity into the dissolution medium in 45 min. A method for cyphetrylin assay in a medium by direct spectrophotometry has been developed. Evaluation of the cyphetrylin release profile showed its gradual release from the tablets, which can be described by a linear relationship in accordance with the Higuchi equation.

Conclusion. As a result of the research, a test Dissolution procedure has been developed and the release profile of cyphetrylin from tablets, 60 mg, was assessed. Experimental data showed that in the selected conditions cyphetrylin release from the tablets is more than 80 % and meets the requirements of the Russian State Pharmacopoeia XV ed.