In Vitro Cytotoxic Potential and In Vivo Antitumor Effects of NOS/PDK-Inhibitor T1084.

Previously, we showed the antitumor activity of the new NOS/PDK inhibitor T1084 (1-isobutanoyl-2-isopropylisothiourea dichloroacetate). The present study included an assessment of in vitro cytotoxicity against human malignant and normal cells according to the MTT-test and in vivo antitumor effects in solid tumor models in comparison with precursor compounds T1023 (NOS inhibitor; 1-isobutanoyl-2-isopropylisothiourea hydrobromide) and Na-DCA (PDK inhibitor; sodium dichloroacetate), using morphological, histological, and immunohistochemical methods. The effects of T1084 and T1023 on the in vitro survival of normal (MRC-5) and most malignant cells (A375, MFC-7, K562, OAW42, and PC-3) were similar and quantitatively equal. At the same time, melanoma A375 cells showed 2-2.5 times higher sensitivity (IC50: 0.39-0.41 mM) to the cytotoxicity of T1023 and T1084 than other cells. And only HeLa cells showed significantly higher sensitivity to the cytotoxicity of T1084 compared to T1023 (IC50: 0.54 ± 0.03 and 0.81 ± 0.02 mM). Comparative studies of the in vivo antitumor effects of Na-DCA, T1023, and T1084 on CC-5 cervical cancer and B-16 melanoma in mice were conducted with subchronic daily i.p. administration of these agents at an equimolar dose of 0.22 mmol/kg (33.6, 60.0, and 70.7 mg/kg, respectively). Cervical cancer CC-5 fairly quickly evaded the effects of both Na-DCA and T1023. So, from the end of the first week of Na-DCA or T1023 treatment, the tumor growth inhibition (TGI) began to decrease from 40% to an insignificant level by the end of the observation. In contrast, in two independent experiments, CC-5 showed consistently high sensitivity to the action of T1084: a significant antitumor effect with high TGI (43-58%) was registered throughout the observation, without any signs of neoplasia adaptation. The effect of precursor compounds on melanoma B-16 was either minimal (for Na-DCA) or moderate (for T1023) with TGI only 33%, which subsequently decreased by the end of the experiment. In contrast, the effect of T1084 on B-16 was qualitatively more pronounced and steadily increasing; it was accompanied by a 3-fold expansion of necrosis and dystrophy areas, a decrease in proliferation, and increased apoptosis of tumor cells. Morphologically, the T1084 effect was 2-fold superior to the effects of T1023-the TGI index reached 59-62%. This study suggests that the antitumor effects of T1084 develop through the interaction of NOS-dependent and PDK-dependent pathophysiological effects of this NOS/PDK inhibitor. The NOS inhibitory activity of T1084 exerts an anti-angiogenic effect on neoplasia. At the same time, the PDK inhibitory activity of T1084 enhances the cytotoxicity of induced intratumoral hypoxia and suppresses the development of neoplasia adaptation to anti-angiogenic stress. Such properties allow T1084 to overcome tumor resistance and realize a stable synergistic antitumor effect.

Combination of NOS- and PDK-Inhibitory Activity: Possible Way to Enhance Antitumor Effects.

We have previously demonstrated a high antitumor potential of NOS inhibitor T1023 (1-isobutanoyl-2-isopropylisothiourea hydrobromide): antitumor antiangiogenic activity in several animal tumor models and its ability to synergistically enhance the antitumor effects of bevacizumab, cyclophosphamide and γ-radiation. At the same time, rather rapid adaptation of experimental neoplasias to T1023 treatment was often observed. We attempted to enhance the antitumor activity of this NOS inhibitor by supplementing its molecular structure with a PDK-inhibiting fragment, dichloroacetate (DCA), which is capable of hypoxia-oriented toxic effects. We synthesized compound T1084 (1-isobutanoyl-2-isopropylisothiourea dichloroacetate). Its toxic properties, NOS-inhibiting and PDK-inhibiting activity in vivo, and antitumor activity on the mouse Ehrlich carcinoma model (SEC) were investigated in compare with T1023 and Na-DCA. We found that the change of the salt-forming acid from HBr to DCA does not increase the toxicity of 1-isobutanoyl-2-isopropylisothiourea salts, but significantly expands the biochemical and anti-tumor activity. New compound T1084 realizes in vivo NOS-inhibiting and PDK-inhibiting activity, quantitatively, at the level of the previous compounds, T1023 and Na-DCA. In two independent experiments on SEC model, a pronounced synergistic antitumor effect of T1084 was observed in compare with T1023 and Na-DCA at equimolar doses. There were no signs of SEC adaptation to T1084 treatment, while experimental neoplasia rapidly desensitized to the separate treatment of both T1023 and Na-DCA. The totality of the data obtained indicates that the combination of antiangiogenic and hypoxia-oriented toxic effects (in this case, within the molecular structure of the active substance) can increase the antitumor effect and suppress the development of hypoxic resistance of neoplasias. In general, the proposed approach can be used for the design of new anticancer agents.

The Ability of the Nitric Oxide Synthases Inhibitor T1023 to Selectively Protect the Non-Malignant Tissues.

Previously, we showed that a nitric oxide synthase (NOS) inhibitor, compound T1023, induces transient hypoxia and prevents acute radiation syndrome (ARS) in mice. Significant efficacy (according to various tests, dose modifying factor (DMF)-1.6-1.9 against H-ARS/G-ARS) and safety in radioprotective doses (1/5-1/4 LD10) became the reason for testing its ability to prevent complications of tumor radiation therapy (RT). Research methods included studying T1023 effects on skin acute radiation reactions (RSR) in rats and mice without tumors and in tumor-bearing animals. The effects were evaluated using clinical, morphological and histological techniques as well as RTOG classification. T1023 administration prior to irradiation significantly limited the severity of acute RSR. This was due to a decrease in radiation alteration of the skin and underlying tissues, and the preservation of the functional activity of cell populations that are critical in the pathogenesis of radiation burn. The DMF values for T1023 for skin protection were 1.4-1.7. Moreover, its radioprotective effect was fully selective to normal tissues in RT models of solid tumors-T1023 reduced the severity of acute RSR and did not modify the antitumor effects of γ-radiation. The results indicate that T1023 can selectively protect the non-malignant tissues against γ-radiation due to hypoxic mechanism of action and potentiate opportunities of NOS inhibitors in RT complications prevention.

Radioprotective Activity of the Nitric Oxide Synthase Inhibitor T1023. Toxicological and Biochemical Properties, Cardiovascular and Radioprotective Effects.

In this work, studies were performed to investigate the toxicological, biochemical, vasotropic and radiomodifying properties of the new nitric oxide synthase (NOS) inhibitor, compound T1023. Toxicological studies included the estimation of acute toxicity in mice after i.p. administration of T1023. Radiometric analysis and electron paramagnetic resonance spectroscopy were used to study NOS-inhibitory properties of T1023 in vitro and in vivo, respectively. T1023 vasoactive properties were studied in rat central hemodynamics. Radiobiological experiments were performed using endogenous and exogenous spleen colony formation as well as 30-day survival tests. The morphological changes in peripheral blood and bone marrow (BM) induced with T1023 were analyzed in mice during hematopoietic acute radiation syndrome (H-ARS). It was shown that T1023 is a sufficiently safe compound (LD10 of 317 mg/kg; LD50 of 410 mg/kg). It is an effective competitive NOS-inhibitor that is 10-to-15-fold selective to endothelial and inducible NOS (IC50 for nNOS, iNOS, eNOS: 52.3, 3.2 and 5.1 µM, respectively). Its NOS-inhibitory activity is realized in vivo and is accompanied by an increase in vascular tone. Its single i.p. administration in doses greater than 1/8 LD10 provides significant (40-50%) and long-lasting (more than 90 min) weakening of cardiac output, which can cause transient hypoxia. In radiobiological studies, T1023 proved to be a hypoxic radioprotector. Its radioprotective effect was observed only when administered prophylactically [single i.p dose, 5-120 min before total-body irradiation (TBI)] and only in doses that reduced cardiac output (1/8 LD10 and more, 40 mg/kg for mice), and was correlated in time with the dynamics of circulatory depression. Its radioprotective effect was not observed when administered in vitro and in the first 4 h after TBI. The optimal radioprotective doses of T1023 are relatively safe (1/ 5-1/4 LD10). In addition, T1023 effectively prevents H-ARS and gastrointestinal acute radiation syndrome (G-ARS) in experimental animals in vivo: dose modifying factor of 1.6-1.9. In the H-ARS mouse model, the prophylactic effect of T1023 (75 mg/kg, single i.p. injection) was accompanied by clinically significant effects. There was an express decrease in the degree of indicators of early BM devastation (by 40%) and maximal neutropenia and thrombocytopenia (2-2.5 times), in addition to a reduction in recovery time (by 30-40%). The obtained experimental results and literature data indicate that NOS inhibitors are an independent class of vasoactive radioprotectors with a specific hypoxic mechanism of action. NOS inhibitors provide new opportunities for developing effective and safe tools for the prevention of ARS.