This work presents the method of synthesis and physicochemical characterization of isothiourea and cinnamic acid original derivative α-cyano-4-hydroxycinnamate 1-cyclohexanoy-l-2-ethylisothiourea (T1114). In studies of the cytotoxic and antitumor activity of T1114, it has been found that the combination in one molecular structure of NOS-inhibitory fragment (1-cyclohexanoyl-2-ethylisothiourea) and a fragment inhibiting monocarboxylate lactate transporters (MCT) (α-cyano-4-hydroxycinnamic acid) does not modify the cytotoxic activity of bifunctional NOS/MCT-inhibitor T1114 in vitro. But in vivo inhibition of NOS and MCT is able to realize effects on the tumor microenvironment and hypoxic tumor cells. Such structural and functional modification has significantly extended the antitumor activity of the new NOS/MCT inhibitor. The bifunctional compound not only realized a more pronounced antitumor effect, but also prevented the development of hypoxic adaptation in solid Ehrlich carcinoma and acquired the ability to overcome the resistance of mouse cervical cancer (RShM-5). Therefore, the combination of NOS-inhibitory, anti-vasculogenic and hypoxia-oriented toxic effects can create new opportunities in antiangiogenic therapy of malignant neoplasms.

According to leading experts, the vast arsenal of radioprotective agents available in the world today does not fully meet modern practical needs, both in the field of radiation protection, and in the prevention and treatment of complications of radiotherapy. The purpose of the study was to evaluate the effect of the salt-forming acids type on the radioprotective activity of NOS inhibitor T1023. The chemical part of this study included methods of chemical synthesis, physicochemical and elemental analysis. Pharmacological part – assessment of acute toxicity using V.B. Prozorovsky express method and the study of radioprotective activity using Till and McCulloch method based on the ability of mice hematopoietic cells to form spleen colonies after irradiation. The number of endogenous hematopoietic spleen colonies were assessed on the 8th day after total exposure to gamma-irradiation at a dose of 6 Gy in six independent experiments. As a result of directed chemical synthesis, six new derivatives of T1023 – salts of N-isobutanoyl-S-isopropylisothiourea have been developed, identified and characterized. The results of studying the safety and radioprotective activity of the synthesized compounds showed that changes in the salt-forming acid don’t significantly influence the toxicity: all studied compounds are in the 3rd class of toxicity and hazard. At the same time, it was found that the replacement of the salt-forming acid significantly influenced the severity of the radioprotective effect. For some of these compounds radioprotective efficacy is comparable to or exceeds the efficacy of the initial compound T1023. It is important to note that these new compounds were used in lower, more save doses than T1023. The results suggest promising further development of NOS inhibitors – isothiourea derivatives as radioprotective agents.

The NO synthase inhibitor T1059 (1-cyclohexanoyl-2-ethylisothiourea hydrobromide) in experiments modeling acute hemorrhagic shock with a single parenteral injection at nontoxic doses (i.p., 10 mg/kg; i.m., 30 mg/kg) showed pronounced hypertensive activity that was much longer than that of phenylephrine and reduced short-term mortality of experimental animals. The results indicated that further pharmacological development of T1059 was promising to design an original domestic drug with antishock activity that is acceptable at the prehospital treatment stage.

The model of combined radiation-thermal injury (CRTI) in mice was experimentally and histologically verified: exposure to 60 Co-radiation at a dose of 7 Gy (0.39 Gy/min) on the Agat device and the subsequent thermal effect on the GL6 device on the back, leading to the development of a 3B degree burn on 10% of the body surface. This model was used to confirm the positive influence of the NOS inhibitor T1023 on the course of CRTI. It was found that a single i.p. administration of T1023 72 h after CRTI application caused a nearly statistically significant (p = 0.051) increase in the 30-day survival rate of animals. The data obtained indicate the need for further detailed study of NOS inhibitor effects on the course of acute radiation syndrome (ARS) and CRTI.

Лактатемия как возможный фармакологический маркер радиорезистентности при действии ингибитора NOS Т1023Макарчук В.М., Филимонова М.В., Филимонов А

The antitumor potential of a new original compound T1097 to realize a complex antineoblastic effect - NOS-inhibiting anti-angiogenic and HK2-inhibiting hypoxia-oriented cytotoxic - was investigated on the model of transplantable Ehrlich carcinoma in vivo. During the entire observation period, T1097 showed pronounced dose-dependent antitumor properties: statistically significant inhibitory effect of T1097 on the growth of tumor nodes exceeded the effect of original compound - HK2-inhibitor 3-bromopyruvate in an equimolar dose. The maximum antineoblastic effect (61%) of T1097 was obtained with its subchronic parenteral administration at a dose of 17 mg/kg and was not accompanied by the development of tumor resistance. Our findings confirm the prospects of this direction in the search for new drugs.

The purpose of the work was to study the ability of the NOS inhibitor T1023 to prevent late radiation injuries. Methods: the effects of T1023 (75 mg / kg, once i.p. 30 minutes before the irradiation) on the development of post-radiation pulmonitis and pneumofibrosis in rats with thoracic exposure to g-radiation at a dose of 12.5 Gy were studied histopathologically and morphometrically. The results of the studies showed that there wasn’t a significant objective effect of T1023 on the development of early radiation-induced lung injuries (9 weeks after irradiation). But it prevented late radiation induced lung injuaries (26 weeks after irradiation) – there were a significant lesser pathomorphological manifestations of post-radiation pulmonitis, proliferation of connective tissue and the development of fibrotic changes in the lung parenchyma. At this stage, the action of T1023 clearly contributed to the preservation of the normal histostructure of the lungs, reducing by 40% the content of compaction zones in the parenchyma. The ability of the NOS inhibitor T1023 to significantly limit the development of lungs late radiation reaction confirms the promise of further development of this compound as a means for prevention radiation therapy complications.

Цель исследования - изучение механизмов противоопухолевой активности ингибитора NOS Т1023 и оценка перспективности его дальнейшей разработки. Методика. В качестве опухолевой модели использована эпидермоидная КЛЛ, штамм которой получен из банка опухолевых материалов ФГБУ РОНЦ им. Н.Н. Блохина и поддерживался на самцах мышей C57BL6j. КЛЛ трансплантировали самцам мышей F1 (CBA´C57BL6j) путем подкожного введения 1,5×106 клеток карциномы в 0,1 мл суспензии на основе среды 199 в область латеральной поверхности правого бедра. Для сравнительной оценки противоопухолевой эффективности использовали ингибитор NOS под шифром Т1023, синтезированный в лаборатории радиационной фармакологии МРНЦ им. А.Ф. Цыба, и VEGF-ингибитор бевацизумаб (БВЗ). Животным первой опытной группы ежедневно, со 2 по 20 сутки вводили соединение Т1023 (60 мг/кг, в/б); второй опытной группы - трижды, на 2, 5 и 10 сут вводили БВЗ (12 мг/кг, в/б); третьей опытной группы - по этим схемам и в таких же дозах вводили и Т1023, и БВЗ (при комбинированном применении Т1023 вводили через 4 ч после введения БВЗ). Контрольным животным в качестве плацебо со 2 по 20 сутки вводили 0,9% раствор натрия хлорида (0,2 мл, в/б). Противоопухолевые эффекты оценивали, сравнивая размеры опухолевых узлов, длительность задержки роста и индекс торможения роста опухоли у контрольных и опытных животных. Гистологические методы исследования включали иммуноокрашивание на PCNA, CD31, пимонидазол и морфометрический анализ микроскопических изображений. Результаты сравнительных исследований показали, что соединение Т1023 и VEGF-ингибитор бевацизумаб (БВЗ) оказывают однонаправленное влияние на карциному легких Льюис (КЛЛ), сопровождающееся торможением роста и подавлением метастазирования неоплазии. Воздействие и Т1023, и БВЗ вызывало снижение содержания сосудов в перитуморальных зонах и в «горячих точках» ангиогенеза, усиливало гипоксию паренхимы КЛЛ и стимулировало апоптоз опухолевых клеток. При комбинированном применении Т1023 и БВЗ их антинеопластическая эффективность в отношении ингибирования ангиогенеза и девитализации опухолевых клеток соответствовала аддитивному действию. Заключение. Результаты позволяют предполагать, что основой противоопухолевой активности Т1023 является антиангиогенное действие и свидетельствуют о перспективности применения ингибиторов NOS в ангиостатической терапии солидных злокачественных новообразований в сочетании с имеющимися антинеоваскулярными средствами. The aim. Study of mechanisms of NOS inhibitor T1023 antitumor activity and estimation of its prospects for further development. Methods. Epidermoid Lewis lung carcinoma (LLC) from N.N. Blokhin NMRCO bank of tumor materials was used as a tumor model. Maintenance of tumor cell culture was provided by intramuscular injection of tumor cells suspension to C57BL6j mice every 14 days. Then LLC cells were transplanted to male F1 mice (CBA´C57BL6j) by subcutaneous injection of 1,5×106 cells in 0,1 ml of 199 medium into the lateral surface of the right hip. Comparative studies of antitumor efficacy were carried out using NOS inhibitor T1023, synthesized in the laboratory of radiation pharmacology of A.F. Tsyb MRRC, and VEGF inhibitor Bevacizumab (BVZ). Mice from the first experimental group were injected intraperitoneally (ip) with compound T1023 at dose 60 mg / kg from day 2 to 20; animals from the second experimental group were treated with BVZ at dose 12 mg / kg ip at days 2, 5 and 10; the third experimental group received T1023 in combination with BVZ according to these schemes and at the same doses (T1023 was administered 4 hours after administration of BVZ). Mice from the control group received 0,9% sodium chloride solution (0,2 ml, ip) as a placebo daily from 2 to 20 days. Antitumor effects were assessed by comparing the tumor size, duration of tumor growth delay and the index of tumor growth inhibition in control and experimental groups. Histological examination methods included immunostaining on PCNA, CD31, pimonidazole and morphometric analysis of microscopic images. Results. Comparative studies have shown that compound T1023 and VEGF inhibitor Bevacizumab (BVZ) have unidirectional effects on Lewis lung carcinoma (LLC), accompanied by growth inhibition and suppression of metastasis of neoplasia. The effect of both T1023 and BVZ caused a decrease in vascular content in the peritumoral zones and in the “hot spots” of angiogenesis, increased the hypoxia in the LLC parenchyma, and stimulated apoptosis of tumor cells. The combined use of T1023 and BVZ, caused the antineoplastic efficacy against inhibition of angiogenesis and devitalization of tumor cells which was estimated as additive effect. Conclusion. The results suggest that the basis of antitumor activity of T1023 is the anti-angiogenic effect and indicate the prospects of using NOS inhibitors in the angiostatic therapy of solid malignant neoplasms in combination with available anti-neovascular agents.

A correlation between the effectiveness of the radioprotective action of cysteamine with the radiosensitivity of cells and their ability to recover from radiation damage was demonstrated for diploid yeast cells of S. cerevisiae. To demonstrate the involvement of the recovery processes in the mechanism for radioprotective action of the T1023 compound in animals, data on the survival of clonogenic bone marrow cells of mice exposed to ionizing radiation are presented. It is shown that the radioprotective effect of this compound can be realized not only at the physicochemical stage of formation of radiation damage due to a decrease in the oxygen content in tissues but also due to the post-radiation cell recovery at the biochemical stage. This is confirmed by the presence of the sigmoid part of the dose-response curve in the region of low doses of ionizing radiation.

В модельных экспериментах, воспроизводящих острый тяжёлый геморрагический шок, показано, что ингибитор синтаз оксида азота Т1059 (1-циклогексаноил-2-этилизотиомочевины гидробромид) при однократном парентеральном введении в нетоксических дозах (внутрибрюшинно, 10 мг/кг; внутримышечно, 30 мг/кг) оказывает выраженное гипертензивное действие, значительно превосходящее по длительности эффект фенилэфрина, и при этом снижает краткосрочную гибель подопытных животных. Полученные результаты свидетельствуют о перспективности дальнейшей фармакологической разработки соединения Т1059 с целью создания оригинального отечественного лекарственного средства, обладающего противошоковой активностью, приемлемого для применения на догоспитальном этапе лечения.

Цель работы - изучение антинеопластических эффектов при комбинированном воздействии ингибитора NOS Т1023 с γ-излучением и циклофосфамидом. Методика. В работе использован штамм эпидермоидной карциномы легких Льюиса. Выполнено 2 независимых эксперимента: в одном изучали антинеопластические эффекты при раздельном и комбинированном воздействии Т1023 и γ-излучения, в другом - при раздельном и комбинированном воздействии Т1023 и циклофосфамида. Особям 1-й опытной группы в обоих экспериментах с 7-х по 20-е сут роста карциномы ежедневно вводили соединение Т1023 в дозе 60 мг/кг внутрибрюшинно. В первом эксперименте животные 2-й опытной группы на 7-е сут роста неоплазии получали сеанс облучения (доза 5 Гр). Во втором эксперименте животным этой группы на 7-е сут роста неоплазии вводили однократно внутрибрюшинно циклофосфамид в дозе 100 мг/кг в виде 1,0% раствора фармакопейного препарата на основе 0,9% раствора натрия хлорида. Животным 3-й опытной группы в обоих экспериментах проводили соответствующие комбинированные воздействия по этим же схемам и в таких же дозах. Первое введение Т1023 на 7-е сут роста карциномы этим животным проводили через 4 ч после облучения или введения циклофосфамида. Влияние на рост опухоли оценивали по межгрупповым различиям объемов опухолевых узлов, длительности задержки роста и индексу торможения роста опухоли. Влияние на активность метастазирования карциномы оценивали по межгрупповым различиям числа легочных метастазов на 21-е сут роста опухоли и индексу ингибирования метастазирования. Статистическую оценку значимости межгрупповых различий количественных показателей проводили с помощью дисперсионного анализа Краскела-Уоллиса с применением Q-критерия Данна. Результаты. Показано, что воздействие Т1023, как в комбинации с γ-излучением, так и в комбинации с циклофосфамидом, сопровождается статистически значимым подавлением роста и метастазирования карциномы легких Льюис. При этом антинеопластические эффекты обоих комбинированных воздействий соответствовали аддитивному противоопухолевому и антиметастатическому действию ингибитора NOS, γ-излучения и циклофосфамида. Заключение. Полученные результаты отражают способность ингибиторов NOS повышать эффективность радио- и химиотерапии злокачественных новообразований, что свидетельствуют о перспективности дальнейшей разработки препаратов типа соединения Т1023. The aim was to study antineoplastic effects of a NOS inhibitor, T1023, in combination with γ-irradiation and cyclophosphamide. Methods. Epidermoid Lewis lung carcinoma (LLC) was used as a tumor model. Two independent experiments were performed. In the first experiment, antitumor effects of T1023 and γ-irradiation were studied individually and combined. In the second experiment, antitumor effects of T1023 and cyclophosphamide were studied individually and combined. In both experiments, mice from the first experimental group were daily injected with T1023 60 mg/kg from day 7 to 20. In the first experiment, animals of the second group were treated with γ-irradiation at 5 Gy on day 7 of tumor growth. In the second experiment, mice of the second group were injected with cyclophosphamide 100 mg/kg on day 7 of tumor growth. In both experiments, mice of the third group received a respective combined treatment according to the protocols described above. The first injection of T1023 was performed on day 7 after tumor transplantation at 4 hours after the irradiation or cyclophosphamide treatment. Antitumor effects were assessed by comparing the tumor size, duration of tumor growth delay, and the index of tumor growth inhibition in control and experimental groups. The effect of treatments on metastatic activity of carcinoma was evaluated by the intergroup difference in number of pulmonary metastases inhibition index on day 21 of tumor growth. Statistical significance was determined using the Kruskal-Wallis dispersion analysis with the Dunn Q-test. Results of the study showed that the T1023 treatment both in combination with γ-irradiation and with cyclophosphamide was associated with a significantly greater inhibition of tumor growth and metastasis. The antineoplastic effect of both combinations was consistent with the additive antitumor and antimetastatic effect of the NOS inhibitor, γ-radiation, and cyclophosphamide. Conclusion. The study showed the ability of NOS inhibitors to enhance the effectiveness of radio- and chemotherapy for malignant tumors and suggested a promising outlook for further development of T1023.

Combined chronic treatment of Ehrlich solid carcinoma (EC) with an NOS inhibitor 1-isobutanoyl-2-isopropylisothiourea hydrobromide (T1023) and a PDK1 inhibitor dichloroacetate was accompanied by statistically significant synergetic antitumor effects manifested in a significant and stable suppression of neoplasm growth (by 55-65%). Separate treatment with T1023 and dichloroacetate induced moderate short-term inhibition of tumor growth (by 30-35%) followed by weakening of tumor sensitivity to these substances. These results attest to synergetic antitumor effects NOS inhibitor T1023 and PDK1 inhibitor dichloroacetate producing antiangiogenic and hypoxia-targeted cytotoxic effects, during their combined administration, which allows overcoming the adaptive potential of the tumors.

Противолучевые эффекты ингибитора синтаз оксида азота Т1023 в нормальных и малигнизированных тканяхФилимонова М.В., Самсонова А.С

Compound T1059 (1-cyclohexanecarbonyl-2-ethylisothiourea hydrobromide) was found to be water soluble, moderately toxic (i.p. LD16 and LD50 of 274 and 380 mg/kg), and capable of competitively inhibiting nitric-oxide synthase (NOS) activity with significant selectivity toward inducible and endothelial isoforms (IC50 for nNOS, iNOS, and eNOS of 60.3, 1.8, and 3.2 μM, respectively). T1059 was rapidly absorbed after a single i.p. injection (dose range 10 – 30 mg/kg) and distributed in tissues, causing pronounced suppression of endogenous NO production. T1059 at a dose of 10 mg/kg in normotensive anesthetized Wistar rats produced long-term vasoconstriction. The observed changes in vascular tone did not influence inotropic heart function but were accompanied by weak bradycardia.