Analytical and metabolic studies of the trypanocidal phenanthridines

A selective, reproducible and precise method for the determination of isometamidium chloride hydrochloride (ISM), in the presence of four process-related and degradation impurities, by RP-HPLC is described for the first time. An HPTLC system with UV densitometric evaluation, suitable for the separation and detection of these compounds is reported also. The Berthelot (Indophenol) reaction was modified for the quantitative determination of an inorganic impurity, ammonium chloride, in commercial samples of ISM (Samorin® and Veridium®). Application of the RP-HPLC method and the modified Berthelot reaction, for the determination of ISM and ammonium chloride respectively, to the analysis of preparations of ISM from different manufacturing sources demonstrated that there are marked qualitative and quantitative differences. Confocal laser scanning microscopy (CLSM) showed that ethidium bromide (EBr) and ISM were taken up rapidly into (the nucleoli and nuclear membranes of isolated rat hepatocytes. The use of HPLC and LC-MS established that EBr was biotransformed to at least six metabolites, by A-glucuronosylation (representing the major pathway), A-acetylation and also by Oglucuronosylation. In rats, the glucuronosylation pathways were differentially induced by phenobarbitone and 3-methylcholanthrene. Metabolites of ISM were not detected; however, its positional isomer (M&B4250, which possesses an unsubstituted 8-amino group; similar to that in EBr) was metabolised by A-glucuronosylation and N-acetylation. Major interspecies variability was observed in the nature and the extent of the metabolism of EBr by rat, sheep and pig hepatocytes. A-glucuronosylation was the major pathway in all three species; with the A-acetylation pathway absent in sheep and pigs.

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