Investigation of biotransformation processes and pharmacological activity of violuric acids and their metabolites in in vivo experiments

Introduction. 5-Hydroxyiminobarbituric (violuric) acid and its derivatives exhibit pronounced anti-hypoxic, hepatoprotective, cytoprotective, actoprotective, and other properties, making this group of compounds a promising field for pharmaceutical research. Data on the metabolism of violuric acids (VAs) are of significant practical and theoretical importance for the tracking of pharmacokinetics and substance distribution within the organism, in which process metabolites serve as markers of biochemical processes involving endogenous substrates.

Objective. To determine the structure of violuric acid metabolites and perform their quantitative assessment in an in vivo experiment.

Materials and methods. The studied substances (VAs) and their metabolites (purpuric acids) were synthesized at the Golikov Research Center of Toxicology. Their structure and purity were confirmed by nuclear magnetic resonance (NMR) spectroscopy, high-performance liquid chromatography (HPLC), and spectrophotometry (SP). Metabolism and anti-hypoxic activity were studied using a model of hemic hypoxia induced by a lethal dose of sodium nitrite in outbred male white rats. Solutions of violuric acids for in vitro studies and administration to animals were prepared in distilled water with the addition of tris(oxymethyl)aminomethane (TRIS), while purpurates in the form of salts were dissolved in distilled water. The dosages of the test substances for rats ranged from 50–75 mg/kg for intraperitoneal administration and 50–100 mg/ kg for intragastric administration. The reference substance (amtizole succinate) was administered to animals at a dose of 100 mg/kg. A 0.9% sodium chloride solution, administered at a volume of 1 mL per animal, was used as a control. Quantitative analysis of the substances and their metabolites in biological media was performed by HPLC with SP detection.

Results. It was established that the studied substances (violuric acid, 2-thiovioluric acid, 1-butylvioluric acid, and 1-(4-bromophenyl)violuric acid) are metabolized in the animal organism to form the corresponding derivatives of purpuric acid (purpurates), whose structure was confirmed by counter synthesis. Violuric acids and their metabolites are primarily excreted in the urine. It was demonstrated that 1-butylvioluric acid and its metabolite N,N’-dibutylpurpuric acid exhibit pronounced anti-hypoxic activity in the model of acute sodium nitrite poisoning, preventing mortality in 100% of animals, whereas the reference antihypoxant amtizole only prolongs survival time (by 23%).

Conclusions. The formation of purpurates is a characteristic pathway of metabolic transformation for the violuric acid scaffold. These metabolites exhibit pronounced activity and, in all likelihood, can contribute to the overall biological effect of violuric acids.

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