Metabolic N-hydroxylation of pentamidine in vitro.

By using high-performance liquid chromatography, the in vitro conversion of pentamidine to the corresponding amidoximes (N-hydroxypentamidine and N,N'-dihydroxypentamidine) was studied in supernatants of rat liver homogenate centrifuged at 9,000 x g. The presence of the two amidoxime peaks in chromatograms was confirmed by liquid secondary ion mass spectrometry and by unequivocal synthesis of the suspected metabolites. The metabolic reactions were found to be catalyzed by the cytochrome P-450 system (mixed-function oxidases). The formation of the monohydroxylated product was found to have a Km of 0.48 mM and a Vmax of 29.50 pmol/min per mg of protein, while the dihydroxylated metabolite had a Km of 0.73 mM and a Vmax of 4.10 pmol/min per mg of protein. N,N'-Dihydroxypentamidine was found to have highly reduced antiprotozoal activity in vitro relative to that of pentamidine, and neither of the hydroxylated metabolites nor pentamidine was found to be significantly mutagenic by the Ames test. Contrary to previous reports, pentamidine is readily metabolized to at least two hydroxylated products, and this conversion may be relevant to the clinical use of the compound and to future drug design.