Medicinal chemistry approaches to avoid aldehyde oxidase metabolism

Aldehyde oxidase (AO) is a molybdenum-containing enzyme distributed throughout the animal kingdom and capable of metabolising a wide range of aldehydes and N-heterocyclic compounds. Although metabolism by this enzyme in man is recognised to have significant clinical impact where human AO activity was not predicted by screening in preclinical species, there is very little reported literature offering real examples where drug discoverers have successfully designed away from AO oxidation. This article reports on some strategies adopted in the Pfizer TLR7 agonist programme to successfully switch off AO metabolism that was seen principally in the rat.     

Oxime-metabolizing activity of liver aldehyde oxidase

Liver aldehyde oxidase in the presence of its electron donor exhibited a significant oxime-metabolizing activity toward some different types of oximes under anaerobic conditions. Acetophenone oxime and salicylaldoxime were exclusively converted to the corresponding oxo compounds, whereas benzamidoxime was converted to the corresponding ketimine. With d-camphor oxime, the formation of both the corresponding oxo compound and ketimine was observed. Stoichiometric studies showed that the formation of oxo compounds is accompanied by nearly equimolar ammonia. We propose a mechanism of oxime biotransformation that liver aldehyde oxidase catalyzes the reduction of oximes to the corresponding ketimines which in turn undergo, depending on their chemical stability, nonenzymatic hydrolysis to the corresponding oxo compounds and ammonia.     

Sulfoxide reductase activity of liver aldehyde oxidase

The present study provides evidence that guinea pig and rabbit liver aldehyde oxidase (EC 1.2.3.1) in the presence of its electron donors such as aldehydes or N-heterocyclic compounds functions as a sulfoxide reductase towards sulindac and other sulfoxide compounds. In addition, the study shows that a combination of liver aldehyde oxidase and milk xanthine oxidase also exhibits sulfoxide reductase activity in the presence of xanthine, and electron donor of xanthine oxidase. Based on these facts, we propose a new electron-transfer system consisting of these two flavoenzymes.     

Chromate reduction by rabbit liver aldehyde oxidase

Chromate was reduced during the oxidation of 1-methylnicotinamide chloride by partially purified rabbit liver aldehyde oxidase. In addition to 1-methylnicotinamide, several other electron donor substrates for aldehyde oxidase were able to support the enzymatic chromate reduction. The reduction required the presence of both enzyme and the electron donor substrate. The rate of the chromate reduction was retarded by inhibitors of aldehyde oxidase but was not affected by substrates or inhibitors of xanthine oxidase. These results are consistent with the involvement of aldehyde oxidase in the reduction of chromate by rabbit liver cytosolic enzyme preparations.     

Side-chain metabolism of propranolol: involvement of monoamine oxidase and aldehyde reductase in the metabolism of N-desisopropylpropranolol to propranolol glycol in rat liver

The further metabolism of N-desisopropylpropranolol (NDP), a side-chain metabolite of propranolol (PL), was investigated in isolated rat hepatocytes. Propranolol glycol (PGL) was generated from NDP as a major metabolite. Naphtetrazole (NTE), a potent inhibitor of monoamine oxidase (MAO), significantly retarded the disappearance of NDP from the incubation medium, suggesting the involvement of MAO in the deamination of NDP to an aldehyde intermediate. In a reaction mixture of rat liver mitochondria and cytosol with NADPH, phenobarbital, a specific inhibitor of aldehyde reductase, and 4-nitrobenzaldehyde (4-NBA), a substrate inhibitor of aldehyde reductase, decreased the formation of PGL from NDP. 4-NBA was a competitive inhibitor of the enzyme responsible for the PGL formation. The optimal pH for the formation of PGL from NDP in the reaction mixture was approximately 8.0. Based on these results, we propose the possibility that, in the rat liver, MAO catalyzes the oxidative deamination of NDP to an aldehyde intermediate and the formed aldehyde intermediate is subsequently reduced to PGL by aldehyde reductase. Furthermore, the enantioselective metabolism of NDP to PGL was examined. In isolated rat hepatocytes, the amount of PGL formed from S-NDP [S(-)-form of NDP] was larger than that of PGL formed from R-NDP [R(+)-form of NDP].     

Purification and immobilization of rabbit liver aldehyde oxidase

Aldehyde oxidase (E.C. 1.2.3.1) was isolated from rabbit liver and two potential bioaffinity ligands, i.e., 3-aminocarbonyl-1-benzyl-6-methylpyridinium bromide and 3-aminocarbonyl-1-benzyl-4,6-dimethylpyridinium chloride, were tested for their applicability in a purification procedure for this enzyme. Various supports and different coupling methods were investigated for the immobilization of aldehyde oxidase. Adsorption to n-hexyl- and n-octylamine-substituted Sepharose 4B and DEAE Sepharose 6B gave the best retention of aldehyde oxidase activity. The storage stability of free enzyme and enzyme immobilized to n-octylamine-substituted Sepharose 4B was studied in several buffers at pH 7.8 and 9.0. This showed that the stability of immobilized enzyme was much less than that of free enzyme. The apparent operational stability of the immobilized enzyme preparation, however, improved substantially compared to soluble enzyme, although the corresponding product yield is still very poor. Coimmobilization of catalase and/or superoxide dismutase provided no significant increase of the apparent operational stability and product yield. A positive effect on both parameters was found for aldehyde oxidase-n-alkylamine Sepharose 4B preparations by increasing the amount of enzyme adsorbed per unit weight of support, whereas the productivity of these preparations remained about constant.     

Side-chain metabolism of propranolol: involvement of monoamine oxidase and mitochondrial aldehyde dehydrogenase in the metabolism of N-desisopropylpropranolol to naphthoxylactic acid in rat liver

We examined the metabolism of N-desisopropylpropranolol (NDP), which is generated from propranolol (PL) by side-chain N-desisopropylation, to naphthoxylactic acid (NLA) in rat liver. S(-)-NDP (S-NDP) and R(+)-NDP (R-NDP) were enantioselectively metabolized to NLA in isolated rat hepatocytes and in an enzyme reaction system of rat liver mitochondria with cofactor NAD+. Furthermore, the clearance profiles of NDP enantiomers were examined in an enzyme reaction system of rat liver mitochondria without NAD+. The amounts of S-NDP remaining in the incubation medium were similar to those of R-NDP, suggesting that monoamine oxidase (MAO) catalyzes the deamination of NDP to the aldehyde intermediate, but fails to deaminate enantioselectively S-NDP or R-NDP. Cyanamide, a potent inhibitor of aldehyde dehydrogenase (ALDH), markedly decreased the formation of NLA from racemic NDP in the enzyme reaction system of rat liver mitochondria with NAD+. When rat liver cytosol and microsomes were added to this enzyme reaction system, no significant alterations were observed in the amount of NLA generated from racemic NDP. We concluded that MAO deaminates NDP to an aldehyde intermediate, and that mitochondrial ALDH subsequently catalyzes the enantioselective metabolism of the aldehyde intermediate to NLA in rat liver.     

The mouse liver aldehyde oxidase locus (Aox)

Wide variability has been demonstrated in the properties and presumably the genetic constitution of aldehyde oxidases of 30 different strains of inbred mice. Genetic control of aldehyde oxidase (Aox) has been shown to reside in linkage group XIII and to be 9.6±0.4 recombination units from isocitric dehydrogenase (Id-1) and 28.3±3.5 recombination units from dipeptidase-1 (Dip-1). On the basis of these data and a recombination percent of 23.5±3.9 for Id-1 and Dip-1, the following gene order was deduced: Aox-Id-1-Dip-1. Furthermore, aldehyde oxidase activity was shown to be independent of adrenal influence and to have no clear-cut survival value for animals treated with large doses of N ¹-methylnicotinamide.This investigation was supported by USPHS grant AM 05741 and by a grant-in-aid from the American Heart Association.     

Involvement of liver aldehyde oxidase in the reduction of nicotinamide N-oxide

The present paper describes that mammalian liver aldehyde oxidase is involved in the reduction of nicotinamide N-oxide to nicotinamide. Rabbit liver aldehyde oxidase supplemented with its electron donor exhibited a significant nicotinamide N-oxide reductase activity under anaerobic conditions. Liver cytosols from rabbits, hogs, guinea pigs, hamsters, rats and mice, all of them, similarly exhibited the N-oxide reductase activity in the presence of an electron donor of aldehyde oxidase, but not xanthine oxidase. The cytosolic N-oxide reductase activity was almost completely inhibited by menadione, an inhibitor of aldehyde oxidase.     

Thin-layer chromatography of hydroxamic acids

The separation of very short-chain from long-chain fatty acyl hydroxamates by thin-layer chromatography is described. The detection limit was 2 micrograms.     

Cynomolgus monkey liver aldehyde oxidase: extremely high oxidase activity and an attempt at purification

Aldehyde oxidase (EC 1.2.3.1) in monkey (Macaca fascicularis) liver was characterized. Liver cytosol exhibited extremely high benzaldehyde and phthalazine oxidase activities based on aldehyde oxidase, compared with those of rabbits, rats, mice and guinea pigs. Monkey liver aldehyde oxidase showed broad substrate specificity distinct from that of the enzyme from other mammals. Purified aldehyde oxidase from monkey liver cytosol showed two major bands and two minor bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These bands were also observed in Western blotting analysis using anti-rat aldehyde oxidase. The molecular mass of the enzyme was estimated to be 130-151 kDa by SDS-PAGE, and to be about 285 kDa by HPLC gel filtration. The results suggest that isoforms of aldehyde oxidase exist in monkey livers.