CHARACTERIZATION AND PARTIAL PURIFICATION OF THE RAT AND HUMAN ENZYME SYSTEMS ACTIVE IN THE REDUCTION OFN-HYDROXYMELAGATRAN AND BENZAMIDOXIME

Abstract

ABSTRACT:The enzymic basis for intracellular reduction of N-hydroxylated amidines to their corresponding amidines, and hydroxylamines to their corresponding amines, is unknown. The hydroxylated amidines can be used as prodrug moieties, and an understanding of the enzyme system active in the reduction can contribute to more efficient drug development. In this study, we examined the properties of this enzyme system using benzamidoxime and N-hydroxymelagatran as substrates. In rats and humans, the hepatic enzyme system was localized in mitochondria as well as in microsomes, using preferably NADH as cofactor. Potassium cyanide, N-methylhydroxylamine, p-hydroxymercuribenzoate, and desferrioxamine were efficient inhibitors, whereas typical cytochrome P450 (P450) inhibitors were ineffective. In rats, the highest specific activity was found in liver, adipose tissue, and kidneys, whereas in humans, the specific activity in the preparations of adipose tissue examined was lower. A sex difference was observed in rat liver, where 4-fold higher activity was seen in microsomes from female rats. No gender differences were present in any other tissue investigated. Partial purification of the hepatic system was achieved using polyethylene glycol fractionation followed by Octyl Sepharose chromatography at low detergent concentrations, whereas the enzyme was denatured after complete solubilization. The unique appearance of the enzyme activity in adipose tissue, together with the cyanide sensitivity and the failure of typical P450 inhibitors to impede the reaction, indicates that the enzyme system active in reduction of benzamidoxime and N-hydroxymelagatran formation is not of cytochrome P450 origin, but likely consists of an NADH-dependent electron transfer chain with a cyanide-sensitive protein as the terminal component.

Amidoxime Reductase System Containing Cytochrome b5 Type B (CYB5B) and MOSC2 Is of Importance for Lipid Synthesis in Adipocyte Mitochondria

“…Enzyme Kinetics of the Amidoxime Reductase-The enzyme kinetics of the amidoxime reductase enzyme system were characterized using subcellular fractions isolated from rat and human liver in the absence and presence of the inhibitor potassium cyanide (KCN) (6). The reductase activity observed in the presence of KCN is considered to be the nonspecific reductase activity, especially at higher benzamidoxime concentrations.…”

“…In a previous study we found high amidoxime reductase activity preferentially in outer mitochondrial membranes (OMM) associated with adipose tissue, liver, and kidney (6). Furthermore, the reductase activity was dependent on the cofactor NADH and inhibited by potassium cyanide (KCN), whereas typical CYP inhibitors, such as carbon monoxide, were ineffective (6) indicating that the reductase is unlikely to be a cytochrome P450 as was suggested previously (10).…”

“…Amidoxime Reduction Assay-Amidoxime reductase activity was determined following the benzamidoxime reduction to benzamidine as described previously (6). The amidoxime reductase activity was also monitored by the reduction of the intermediate metabolite of AZD0837, the N-hydroxylated amidine AR-H069927 to the corresponding amidine AR-H067637 (supplemental Fig.…”

“…Furthermore, the reductase activity was dependent on the cofactor NADH and inhibited by potassium cyanide (KCN), whereas typical CYP inhibitors, such as carbon monoxide, were ineffective (6) indicating that the reductase is unlikely to be a cytochrome P450 as was suggested previously (10). Several studies have attempted to identify the third component, and the involvement of stearoyl-CoA desaturase (11) and cytochrome P450 isoenzyme CYP2D (10) in the microsomal fraction as well as a novel molybdenum-containing enzyme in the mitochondrial fraction (12) was suggested.…”

“…Instead, prodrugs have been developed by N-hydroxylation of these amidine groups, converting them into amidoximes to improve their oral bioavailability as exemplified by the direct thrombin inhibitor ximelagatran (1,2), its follow-up compound AZD0837 (3), and a platelet inhibitor sibrafidan (4). Once these prodrugs are absorbed into the body, they are reduced back to their bioactive amidines by a reductive enzyme system believed to be composed of cytochrome b 5 (CYB5), 3 cytochrome b 5 reductase (CYB5R), and a third unknown component and is associated with both the microsomal fraction (5) as well as the mitochondrial fraction (6,7). The reductive metabolism of nitrogen-containing groups is not only of importance for the activation of prodrugs but also for reduction of hydroxylamines and for detoxification of carcinogenic N-hydroxylated aromatic and heterocyclic amines (8,9).…”