Mechanism of energotropic and antioxidant action of Thiotriazolin

I.F. Belenichev, Head of the Pharmacology and Medical Formulation Department; I.A. Mazur, Head of the Pharmaceutical Chemistry Department; M.A. Voloshyn, MD, Head of the Human Anatomy Department, Zaporizhia State Medical University, Farmatron R&D and Production Association, Zaporizhia; N.O. Horchakova, MD; I.S. Chekman, MD, Head of the Pharmacology Department, O.O. Bohomolets National Medical University, Kyiv 

The search for metabolically active drugs, their synthesis and discovery of their mechanisms of action have been of constant interest to clinicians, pharmacologists, pharmacists and biochemists since the end of the last century.

Today, the principal mechanisms of cerebral and myocardial metabolism impairment in ischemic conditions have been established, and the key role of major energy metabolism intermediates in the cellular adaptive responses has been discovered [18, 30, 31, 35]. In addition, a new class of metabolically acting agents was developed based on the substances which activate the endogenous bioenergetic processes of the cell channeling them to a natural physiological pathway, reducing the activity of the radical-initiating reactions and coordinating the citric acid cycle reactions [10, 23, 27, 34, 36].

At the same time, based on current concepts of ischemic injury (uncoordinated reactions in the citric acid cycle, anaerobic glycolysis activation, initiation of the lipid peroxidation (LPO) reactions, inhibition of the antioxidant enzymes), the first original domestic drug Thiotriazolin (morpholinium 3-methyl-1,2,4-triazolyl-5-thioacetate) was developed. This medicinal product immediately took its rightful place among metabolically active drugs with marked antioxidant effects. Thanks to the presence of sulfur, a triazole ring and a methyl group in its chemical structure, Thiotriazolin, as a metabolically-active drug, demonstrates a wide range of pharmacological activity, which is especially relevant in clinical pharmacology. Thiotriazolin shows antioxidant, anti-ischemic, membrane-stabilizing, anti-arrhythmic, immunomodulating, anti-inflammatory, hepato-, cardio-, neuro- and nephroprotective activities [3, 5, 7-9, 22]. Thanks to such a wide range of actions, Thiotriazolin can be used in cardiology, hepatology, gynecology, neurology, pediatrics and surgery in the combination treatment of myocardial infarction, angina pectoris, stroke, hepatitis of various etiology, congenital renal anomalies, etc.

Recent experimental evidence has proved the efficacy of Thiotriazolin in treating various liver injuries (caused by tetracycline and isoniazid-rifampicin, doxorubicin, alcohol, carbon tetrachloride) thanks to its antioxidant effect which allows to prevent cytolysis [13, 14]. Addition of Thiotriazolin to combination therapy regimens in patients with liver cirrhosis promoted restoration of normal LPO and antioxidant protection parameters, while at the same time reducing the severity of neurasthenia, dyspepsia, edema and ascites [21]. In patients with post-infarction angina, Thiotriazolin reduces the sensitivity of the myocardium to adrenergic cardiostimulatory effects of catecholamines, prevents progressive suppression of the contractile function of the heart, restores normal myocardial repolarization, delimits the focal areas of necrosis, reduces the ischemic areas and the number of recurrent infarctions [20]. The product increases the contractile activity of the myocardium and improves regional circulation in patients with chronic heart failure [15].

Protective effect of Thiotriazolin on the course of the chronic autoimmune inflammatory process has been established in experimental clinical trials. Thiotriazolin prevents progressive myopathy and helps maintain muscle mass in rats with adjuvant arthritis. The product allows to prevent muscle mass loss, increase the force of muscle contraction and improve the functional capacity in patients with rheumatoid arthritis [28].

Thiotriazolin is a component of the combination drug Tiocetam which improves the condition of patients with cerebrovascular disease and previous stroke, has favorable effects on mnestic functions, motor performance and cerebral hemodynamics and restores the nucleic acid metabolism parameters to normal values [1, 16]. The combination medicinal product Thiodaron is used to treat different types of arrhythmias.

The mechanism of action of Thiotriazolin, however, remains unclear.

Mechanism of Thiotriazolin effects on energy metabolism

Studies investigating the mechanism of action of Thiotriazolin, conducted in 1983-1995 by medical and biological departments of Zaporizhia State Medical University, showed that the product’s efficacy was based on its ability to reduce the extent of oxidative processes suppression in the citric acid cycle, enhance the compensatory activation of anaerobic glycolysis, increase the intracellular ATP stores (by preserving oxidative energy production in the tricarboxylic part and affecting the activation of the dicarboxylic part) and stabilize cell metabolism [6, 19].

A number of important properties of Thiotriazolin have been established in experimental in vivo and in vitro models, including its low toxicity, high cytoprotective activity irrespective of the cell type (cardiomyocytes, hepatocytes, neurocytes, etc.) and modulating effect in normal and pathological conditions [19, 22], which suggests the versatility of product effects.

In the setting of ischemic tissue injury, Thiotriazolin restores normal utilization of cellular glucose and glycogen stores and glucose-6-phosphate dehydrogenase activity, increases the NAD/NADH ratio and cytochrome C oxidase activity, as well as the levels of pyruvate, malate, isocitrate and succinate. In addition, it reduces lactate overproduction and manifestations of uncompensated acidosis and its pro-oxidant action (Fig. 1). Intensification of the oxidative carbohydrate metabolism by Thiotriazolin promotes an increase in the ATP content associated with increased ADP stores and (which is of crucial importance) a decrease in the AMP level [2, 9].

Thiotriazolin activates NADH-dependent oxidation of ethanol in mitochondria which play the key role in energy supply to the cell [4, 22]. Apparently, Thiotriazolin helps utilize reduced pyridine nucleotides in the mitochondrial malate-aspartate shunt (Fig. 1) and activates NADH oxidation in the cytosolic lactate dehydrogenase reaction during the development of ischemia. Promoting the utilization of reduced pyridine nucleotides (Fig. 1), Thiotriazolin inhibits production of reactive oxygen species (ROS) and activates oxidative phosphorylation with increased ATP synthesis. The protective effect of Thiotriazolin in ischemia is probably realized by activating the malate-aspartate shuttle mechanism which supplies protons to the electron transport chain. Compensatory increase in the intensity of the malate shunt, in turn, is accompanied by inhibition of the carbohydrate conversion to acetyl-CoA (in the pyruvate dehydrogenase reaction) which has an effect on free fatty acid synthesis in ischemia. Activation of the malate-aspartate mechanism by Thiotriazolin not only promotes ATP production but helps inhibit pathological lipid synthesis [2, 19, 22].

In ischemic cells and tissues, Thiotriazolin considerably inhibits free amino acid accumulation, increases the RNA level and activates the process of protein synthesis, which suggests the initiation of the cellular adaptive responses resulting in tissue metabolism alteration in hypoxic conditions without increasing the oxygen demand and without free radical production [7, 19, 22].

Therefore, Thiotriazolin exerts its effect on oxidative energy production in the setting of ischemia by activating the malate-aspartate shunt, supplying protons to the electron transport chain, increasing the utilization of reduced pyridine nucleotides, inhibiting ROS production in bioenergetic reactions, decreasing pathological lipid synthesis and significantly stimulating adaptive protein synthesis.

Mechanism of the antioxidant action of Thiotriazolin

According to some authors, the antioxidant action of Thiotriazolin is attributable to the activation of antioxidant enzymes (such as superoxide dismutase, catalase, glutathione peroxidase), which promotes a more sparing use of the endogenous antioxidant α-tocopherol and inhibits the production of lipid peroxidation intermediates and final products, i.e., conjugated dienes, ketotrienes and malondialdehyde [2-4, 7]. Several works addressing the inhibitory effect of Thiotriazolin on the oxidative modification of proteins have been published recently [19, 22]. The discovery of the product’s considerable antioxidant activity exerted at initial stages of free-radical oxidation and permanently observed in different pathological process models [22] allowed us to assume that the ROS scavenging ability is involved in the mechanism of the antioxidant action of Thiotriazolin. Experiments іn vіvо have shown that the product used in the concentration range of 10-5 to 10-7 М reduces concentrations of certain ROS, such as superoxide radical (О2-) and peroxynitrite (ОNОО-) [3, 6, 7]. This effect of Thiotriazolin is due to the presence of a thiol group in its structure, which confers to the molecule its highly reducing properties and the ability to accept electrons from ROS with a change of the sulfur valence number in the thiol group from 2 to 4 (Fig. 2).

Thiotriazolin not only scavenges ROS thanks to the reducing properties of the thiol group (Fig. 2), but inhibits major ways of their production; it reduces ROS production in mitochondria by utilizing reduced pyridine nucleotides and maintaining oxidative energy production [22] and acts on the same process in the xanthine oxidase reaction both by restoring normal metabolism of adenyl nucleotides and inhibiting xanthine dehydrogenase oxidation to xanthine oxidase caused by ROS [4]. Apparently, Thiotriazolin limits ROS production in mitochondria thanks to its direct inhibitory effect on mitochondrial NADH oxidase, which was established in our in vitro studies [5], and reduces the activating effect of metabolic acidosis on ROS-generating systems. The product prevents oxidative modification of the protein structures of receptors, ion channels, enzymes, transcription factors, etc., by reducing the overproduction of superoxide radical and peroxynitrite.

The protective action of Thiotriazolin has been studied most extensively on sulfhydryl groups of cysteine and methionine moieties in protein molecules. The drug competes with these structures for superoxide radical and thus prevents their reversible or irreversible modification. The inhibition of reversible modification prevents the formation of –S–S– bonds in cysteine-containing regions of Nа+/К+-ATPase and reduces the loss of enzyme sensitivity to the regulatory effect of ATP. Reduced formation of –S–S– bonds in the xanthine dehydrogenase molecule caused by Thiotriazolin prevents its conversion to xanthine oxidase and the production of ROS.

Thiotriazolin acts more efficiently on the irreversible modification of sulfhydryl groups of some protein molecules caused by ROS. It inhibits the formation of the readily oxidizable irreversible sulfoxides and sulfonic groups in proteins. By inhibiting the irreversible oxidative modification of sulfhydryl groups in cysteine moieties of protein molecules, Thiotriazolin restores the balance of redox regulation in oxidative stress conditions (Fig. 2). The product prevents imbalance of the thiosulfide system in the event of ROS overproduction and mediates such functions as cell signal transduction via a receptor-ionophore complex, maintenance of the protein, enzyme and transcription factor activities and cell membrane integrity [22]. In the in vitro models of oxidative and nitrosative stress induced by Fenton’s reagent and excess nitroprusside, Thiotriazolin in the concentrations of 10-5 to 10-7 М was shown to prevent cysteine oxidation and cysteine sulfoxide formation and suppress nitrotyrosine formation [4, 22]. Therefore, it can be concluded that Thiotriazolin prevents irreversible inactivation of the NF-kappaB transcription factor, protecting the sensitive cysteine residues Cys 252, Cys 154 and Cys 61 in its DNA-binding domains from excess ROS (Fig. 2). Moreover, Thiotriazolin can be involved in reduction of these groups during reversible inactivation, taking the role of a redox factor.

By inhibiting the oxidative inactivation of NF-kappaB transcription factor in the setting of excess ROS, Thiotriazolin arguably enhances the activation of expression of the redox-sensitive genes required to protect cells from the toxic effects of oxidative stress [18, 31-35]. Some of these genes are responsible for superoxide dismutase synthesis [5, 6, 9, 19, 22], which supports the effect of Thiotriazolin on the increase in superoxide dismutase activity in the setting of ischemia and other extreme states of the human body. Another mechanism for increasing the activity of the said enzyme may be the direct protection of the SOD metalloprotein complex containing copper, zinc or manganese from excess peroxynitrite by Thiotriazolin (Fig. 2).

Thiotriazolin has recently been shown to exert an anti-apoptotic effect [4, 22]. We were the first to anticipate the potential close relationship between the said effect and the antioxidant action of the product. The  property of Thiotriazolin to maintain thiosulfide balance both by directly competing with sulfhydryl compounds for superoxide radical and peroxynitrite and by activating the glutathione peroxidase/reductase system as described in our previous works [5, 6, 22] is likely to help maintain the balance in the pair “oxidized thioredoxin – thioredoxin reduced in the setting of excess ROS” (Fig. 2). The product reduces the accumulation of excess oxidized thioredoxin and probably inhibits the proapoptotic JNK MAP-kinase pathway, thus reducing the initiation of apoptosis.

The mechanism of cardioprotective action of Thiotriazolin in doxorubicin cardiomyopathy has been studied in the context of the ability of thiol groups to prevent toxic effects of anthracycline compounds (thanks to their antioxidant activity) and disorders of the oxidative phosphorylation and mitochondrial respiration rate [17]. Thiotriazolin prevents cardiac and systemic hemodynamics disorders developing during doxorubicin administration in rabbits [25] and prevents intensification of LPO processes and inhibition of the antioxidant protection enzymes [26]. The membrane protection properties of the product are related to the structure of polyunsaturated fatty acids. Thiotriazolin administered concomitantly with doxorubicin prevented a decrease in the linoleic acid level and an increase in the arachidonic acid level in the rat myocardium and a decrease in the polyunsaturated fatty acid levels, an increase in the arachidonic acid level and a change in the ratio of saturated to unsaturated fatty acids in rat liver tissues. The obtained results demonstrate that a potent inducing effect of Thiotriazolin on the antioxidant protection system inhibits overproduction of the LPO products in pathologically changed tissues, thus ensuring structural and functional integrity of the cell membranes [24].

Therefore, by lowering the ROS (superoxide radical and peroxynitrite) concentrations by direct interaction and inhibition of their production pathways, Thiotriazolin reduces the extent of oxidative modification of certain protein structures (antioxidant enzymes, receptors, enzymes involved in energy reactions), maintains thiosulfide balance in the redox regulation system and enhances the synthesis of compounds (antioxidant enzymes, transcription factors, transport proteins) which increase the cell’s resistance to extreme factors.

Figure 1. Effects of Thiotriazolin on energy metabolism

Figure 2. Mechanisms of the antioxidant action of Thiotriazolin

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