In order to examine the importance of metabolic cycles and in particular of reductions of N-hydroxylated compounds, the reversible metabolism at the amidine, guanidine, and amidinohydrazone nitrogen atoms of various drugs and model compounds was investigated. Many of these N-oxygenated metabolites are very easily reduced back into the starting materials. A comparison of the kinetic data for the N-hydroxylation and reduction suggests that the reduction should predominate in vivo. This could be verified by in vivo studies. Thus, N-hydroxylated amidines (amidoximes) can be used as pro-drugs of amidines.Because of their strong basicity, amidines, guanidines, and amidinohydrazones are protonated under physiological conditions, are very hydrophilic, and are usually not absorbed from the gastrointestinal tract. The N-hydroxylated derivatives of amidines (amidoximes), guanidines (N-hydroxyamidines), and amidinohydrazones (N-hydroxyamidinohydrazones) are less basic because of the introduction of the oxygen atom. T...

NOSs (nitric oxide synthases) catalyse the oxidation of L-arginine to L-citrulline and nitric oxide via the intermediate NOHA (Nω-hydroxy-L-arginine). This intermediate is rapidly converted further, but to a small extent can also be liberated from the active site of NOSs and act as a transportable precursor of nitric oxide or potent physiological inhibitor of arginases. Thus its formation is of enormous importance for the nitric-oxide-generating system. It has also been shown that NOHA is reduced by microsomes and mitochondria to L-arginine. In the present study, we show for the first time that both human isoforms of the newly identified mARC (mitochondrial amidoxime reducing component) enhance the rate of reduction of NOHA, in the presence of NADH cytochrome b5 reductase and cytochrome b5, by more than 500-fold. Consequently, these results provide the first hints that mARC might be involved in mitochondrial NOHA reduction and could be of physiological significance in affecting endogenous nitric oxide levels. Possibly, this reduction represents another regulative mechanism in the complex regulation of nitric oxide biosynthesis, considering a mitochondrial NOS has been identified. Moreover, this reduction is not restricted to NOHA since the analogous arginase inhibitor NHAM (Nω-hydroxy-Nδ-methyl-L-arginine) is also reduced by this system.

N-Hydroxylated amidines (amidoximes) can be used as prodrugs of amidines. The prodrug principle was developed in our laboratory for pentamidine and had been applied to several other drug candidates. One of these compounds is melagatran, a novel, synthetic, low molecular weight, direct thrombin inhibitor. To increase the poor oral bioavailability due to its strong basic amidine functionality selected to fit the arginine side pocket of thrombin, the less basic N-hydroxylated amidine was used in addition to an ethyl ester-protecting residue. The objective of this investigation was to study the reduction and the hydrolytic metabolism of ximelagatran via two mono-prodrugs (N-hydroxy-melagatran and ethyl-melagatran) to melagatran by in vitro experiments. New high-performance liquid chromatography methods were developed to analyze all four compounds. The biotransformation of ximelagatran to melagatran involving the reduction of the amidoxime function and the ester cleavage could be demonstrated in vitro by microsomes and mitochondria from liver and kidney of pig and human, and the kinetic parameters were determined. So far, one enzyme system capable of reducingN-hydroxylated structures has been identified in pig liver microsomes, consisting of cytochromeb₅, NADH-cytochromeb₅ reductase, and a P450 isoenzyme of the subfamily 2D. This enzyme system also reduces ximelagatran andN-hydroxy-melagatran. The participation of recombinant human CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, and 3A4 with cytochromeb₅ and b₅reductase in the reduction can be excluded. In summary, ximelagatran and N-hydroxy-melagatran are easily reduced by several enzyme systems located in microsomes and mitochondria of different organs.

Significance: The involvement of biotransformation enzymes in drug metabolism is one of the most crucial objectives during preclinical research, since they ultimately determine the bioavailability of medicinal drugs. The mARC N-reductive enzyme system was found to be a highly effective counterpart to one of the most prominent biotransformation enzymes, CYP450, and is involved in activation of amidoxime prodrugs as well as inactivation of other drugs containing N-hydroxylated functional groups. Owing to its potent N-reductive capacity toward a broad range of compounds, including mutagenic N-oxygenated nucleobase analogs, mARC plays a crucial role in pharmacology. Our crystal structure of human mARC forms the basis for predictions on the metabolism of drug candidates and structure–activity relationships. Moreover, it indicates the evolutionary development of different molybdoenzyme families.