NOSs (nitric oxide synthases) catalyse the oxidation of L-arginine to L-citrulline and nitric oxide via the intermediate NOHA (N(ω)-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 (N(ω)-hydroxy-N(δ)-methyl-L-arginine) is also reduced by this system.

The recently discovered mammalian molybdoprotein mARC1 is capable of reducing N-hydroxylated compounds. Upon reconstitution with cytochrome b(5) and b(5) reductase, benzamidoxime, pentamidine, and diminazene amidoximes, N-hydroxymelagatran, guanoxabenz, and N-hydroxydebrisoquine are efficiently reduced. These substances are amidoxime/N-hydroxyguanidine prodrugs, leading to improved bioavailability compared to the active amidines/guanidines. Thus, the recombinant enzyme allows prediction about in vivo reduction of N-hydroxylated prodrugs. Furthermore, the prodrug principle is not dependent on cytochrome P450 enzymes.

With the emergence of oseltamivir-resistant influenza viruses and in view of a highly pathogenic flu pandemic, it is important to develop new anti-influenza agents. Here, the development of neuraminidase (NA) inhibitors that were designed to overcome resistance mechanisms along with unfavorable pharmacokinetic (PK) properties is described. Several 5-guanidino- and 5-amidino-based oseltamivir derivatives were synthesized and profiled for their anti-influenza activity and in vitro and in vivo PK properties. Amidine 6 and guanidine 7 were comparably effective against a panel of different A/H1N1 and A/H3N2 strains and also inhibited mutant A/H1N1 neuraminidase. Among different prodrug strategies pursued, a simple amidoxime ethyl ester (9) exhibited a superior PK profile with an oral bioavailability of 31% (rats), which is comparable to oseltamivir (36%). Thus, bioisosteric replacement of the 5-guanidine with an acetamidine-in the form of its N-hydroxy prodrug-successfully tackled the two key limitations of currently used NA inhibitors, as exemplified with oseltamivir.

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