In Vitro Study of 6-mercaptopurine Oxidation Catalysed by Aldehyde Oxidase and Xanthine Oxidase

doi:10.2133/dmpk.22.299

Drug Metabolism and Pharmacokinetics

Volume 22, Issue 4, 2007, Pages 299-306

https://doi.org/10.2133/dmpk.22.299 Get rights and content

Summary:

In spite of over 40 years of clinical use of 6-mercaptopurine, many aspects of complex pharmacology and metabolism of this drug remain unclear. It is thought that 6-mercaptopurine is oxidized to 6-thiouric acid through 6-thioxanthine or 8-oxo-6-mercaptopurine by one of two molybdenum hydroxylases, xanthine oxidase (XO), however, the role of other molybdenum hydroxylase, aldehyde oxidase (AO), in the oxidation of 6-mercaptopurine and possible interactions of AO substrates and inhibitors has not been investigated in more details. In the present study, the role of AO and XO in the oxidation of 6- mercaptopurine has been investigated. 6-Mercaptopurine was incubated with bovine milk xanthine oxidase or partially purified guinea pig liver molybdenum hydroxylase fractions in the absence and presence of XO and AO inhibitor/substrates, and the reactions were monitored by spectrophotometric and HPLC methods. According to the results obtained from the inhibition sudies, it is more likely that 6-mercaptopurine is oxidized to 6-thiouric acid via 6-thioxanthine rather than 8-oxo-6-mercaptopurine. The first step which is the rate limiting step is catalized solely by XO, whereas both XO and AO are involved in the oxidation of 6-thioxanthine to 6-thiouric acid.

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2023, Metalloenzymes: From Bench to Bedside
Molybdenum is an essential and crucial element for the survival of mammalians. Five mammalian Mo-dependent enzymes are known and all of them bearing a molybdenum cofactor (Moco) in the active site. Inside the Moco, molybdenum shuttles between two oxidation states, Mo (IV) and Mo (VI) following substrate reduction or oxidation. In eukaryotes, Moco is synthesized through a highly conserved biosynthetic pathway. A deficiency in the biosynthesis of Moco results in a loss of all four human Mo-enzyme activities and in major cases in early childhood death. In this chapter, we initially introduce general aspects of molybdenum biochemistry, then, we focus on the functions, deficiencies, and presence of known inhibitors/activators of the five Mo-enzymes, xanthine dehydrogenase, sulfite oxidase, aldehyde oxidase, nitrate reductase, and mARC.
Drug Metabolism: Other Phase I Enzymes
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To this date, most of the scientific literature related to drug metabolism has focused on the characterization of cytochrome P450-mediated processes, with the other phase I drug metabolizing enzymes largely overlooked. However, an increasing volume of recent studies have highlighted the important contribution of non-P450 human drug-metabolizing enzymes in converting drugs/pro-drugs into either more hydrophilic compounds, non-toxic excretable metabolites or activated drugs.
Based on these new data, this chapter presents the current state of the art of other Phase I drug metabolizing enzymes by reviewing a selected group of these enzymes. Special attention is devoted to flavin-containing monooxygenases (FMOs), for which new interesting data have become available concerning their structural features, their overall catalytic mechanism, as well as the effect of polymorphisms on their activity.
In addition to FMOs, four other phase I drug metabolizing enzymes are also briefly reviewed including aldehyde oxidases, aldehyde dehydrogenases, alcohol dehydrogenases and carboxylesterases. For each of these enzymes the catalytic mechanism is presented together with information on their specific role in drug metabolism and the available kinetic data for the metabolism of drugs.
The resulting content of this chapter aims at becoming a source of relevant information as well as elaboration of data for these highly relevant enzymes.
Insights into the cellular pharmacokinetics and pharmacodynamics of thiopurine antimetabolites in a model of human intestinal cells
2021, Chemico-Biological Interactions
Citation Excerpt :
However, in our case, IHH and HCEC lines seem to have a similar HPRT expression [42] suggesting no influence by this enzyme in terms of metabolite concentration. Aldehyde oxidase (AO) and xanthine oxidase (XO) contribute to mercaptopurine detoxification through the production of hydroxythioguanine and thiouric acid, respectively [43]. In particular, being AO metabolism highly predominant in hepatocytes [44], we hypothesize that the higher AO activity in hepatocytes could be partially responsible for metabolites reduction after 96 h of thiopurine treatment in IHH cells, in comparison to the constant metabolite levels in HCEC cells.
Thiopurines, immunomodulating drugs used in the management of different chronic autoimmune conditions and as anti-leukemic agents, may exert in some cases gastrointestinal toxicity. Moreover, since these agents are administered orally, they are absorbed across the gastrointestinal tract epithelium. On these premises, cellular and molecular events occurring in intestinal cells may be important to understand thiopurine effects. However, quantitative information on the biotransformation of thiopurines in intestinal tissues is still limited. To shed light on biotransformation processes specific of the intestinal tissue, in this study thiopurine metabolites concentrations were analyzed by an in vitro model of human healthy colon, the HCEC cell line, upon exposure to cytotoxic concentrations of azathioprine or mercaptopurine; the investigation was carried out using an innovative mass spectrometry method, that allowed the simultaneous quantification of 11 mono-, di-, and triphosphate thionucleotides. Among the 11 metabolites evaluated, TIMP, TGMP, TGDP, TGTP, MeTIMP, MeTIDP and MeTITP were detectable in HCEC cells treated with azathioprine or mercaptopurine, considering two different incubation times before the addition of the drugs (4 and 48 h). Different associations between metabolites concentrations and cytotoxicity were detected. In particular, the cytotoxicity was dependent on the TGMP, TGDP, TGTP and MeTITP concentrations after the 4 h incubation before the addition of thiopurines. This may be an indication that, to study the association between thiopurine metabolite concentrations and the cytotoxicity activity in vitro, short growth times before treatment should be used. Moreover, for the first time our findings highlight the strong correlation between cytotoxicity and thiopurine pharmacokinetics in HCEC intestinal cells in vitro suggesting that these cells could be a suitable in vitro model for studying thiopurine intestinal cytotoxicity.
Aldehyde oxidase and its role as a drug metabolizing enzyme
2019, Pharmacology and Therapeutics
Aldehyde oxidase (AO) is a cytosolic enzyme that belongs to the family of structurally related molybdoflavoproteins like xanthine oxidase (XO). The enzyme is characterized by broad substrate specificity and marked species differences. It catalyzes the oxidation of aromatic and aliphatic aldehydes and various heteroaromatic rings as well as reduction of several functional groups. The references to AO and its role in metabolism date back to the 1950s, but the importance of this enzyme in the metabolism of drugs has emerged in the past fifteen years. Several reviews on the role of AO in drug metabolism have been published in the past decade indicative of the growing interest in the enzyme and its influence in drug metabolism. Here, we present a comprehensive monograph of AO as a drug metabolizing enzyme with emphasis on marketed drugs as well as other xenobiotics, as substrates and inhibitors. Although the number of drugs that are primarily metabolized by AO are few, the impact of AO on drug development has been extensive. We also discuss the effect of AO on the systemic exposure and clearance these clinical candidates. The review provides a comprehensive analysis of drug discovery compounds involving AO with the focus on developmental candidates that were reported in the past five years with regards to pharmacokinetics and toxicity. While there is only one known report of AO-mediated clinically relevant drug-drug interaction (DDI), a detailed description of inhibitors and inducers of AO known to date has been presented here and the potential risks associated with DDI. The increasing recognition of the importance of AO has led to significant progress in predicting the site of AO-mediated metabolism using computational methods. Additionally, marked species difference in expression of AO makes it is difficult to predict human clearance with high confidence. The progress made towards developing in vivo, in vitro and in silico approaches for predicting AO metabolism and estimating human clearance of compounds that are metabolized by AO have also been discussed.
Thiopurine-induced toxicity is associated with dysfunction variant of the human molybdenum cofactor sulfurase gene (xanthinuria type II)
2018, Toxicology and Applied Pharmacology
Citation Excerpt :
XDH/XO is a crucial enzyme in the azathioprine catabolic pathway. It catalyzes the oxidation of 6-mercaptopurine to 6-thioxanthine and 6-thiouric acid (Rashidi et al., 2007). Aldehyde oxidase (AOX, EC:1.2.3.1) is a molybdenum-containing oxidoreductase that is similar to XDH/XO and primarily catalyzes the oxidation of aldehydes into carboxylic acids.
The aim of our study was to identify the genetic background of thiopurine-induced toxicity in a patient with a wild-type thiopurine methyltransferase genotype and activity. A 38-year-old Caucasian woman presented with cutaneous necrotizing vasculitis pancytopenia one month after starting azathioprine therapy.
During a routine biochemical follow-up of the patient, undetectable serum uric acid (<10 μl) was observed. A high performance liquid chromatography analysis of urinary purines revealed increased levels of xanthine (137 mmol/mol creatinine). The suspected diagnosis of hereditary xanthinuria, a rare autosomal recessive disorder of the last two steps of purine metabolism, was confirmed by sequence analysis.
An analysis of XDH/XO and AOX1 revealed common polymorphisms, while analysis of the MOCOS gene identified a rare homozygous variant c.362C > T. Dysfunction of this variant was confirmed by significantly decreased xanthine dehydrogenase/oxidase activity in the patient's plasma (<2% of control mean activity).
We present a biochemical, enzymatic, and molecular genetic case study suggesting an important association between a hitherto undescribed dysfunction variant in the MOCOS gene and thiopurine-induced toxicity. The identified variant c.362C > T results in slower thiopurine metabolism caused by inhibition of 6-mercaptopurine oxidation (catabolism) to 6-thioxanthine and 6-thiouric acid, which increases the formation of the nucleotide 6-thioguanine, which is toxic. This is the first clinical case to identify the crucial role of the MOCOS gene in thiopurine intolerance and confirm the impact of genetic variability of purine enzymes on different therapeutic outcomes in patients undergoing thiopurine treatment.
Xanthine Oxidoreductase and Aldehyde Oxidases
2018, Comprehensive Toxicology: Third Edition
Aldehyde oxidases (AOXs) and xanthine oxidoreductase (XOR) are closely related enzymes with very similar primary structures comprising two identical subunits each of which contains four redox centers: one molybdopterin cofactor, one flavin adenine dinucleotide (oxidized form) molecule, and two iron–sulfur clusters. The enzymes, also referred to as molybdenum hydroxylases, catalyze both oxidation and reduction reactions and play a significant role in the metabolism of drugs and endobiotics including endogenous purines. Generally, oxidation reactions involve nucleophilic attack via a Mo–OH ligand at a carbon atom in N-heterocycles and aldehydes with electrons transferred ultimately to molecular oxygen or NAD⁺ (nicotinamide adenine dinucleotide—the oxidized form). Reduction of oxygen can generate reactive oxygen species, which have been implicated in a variety of protective and pathophysiological functions. AOXs and XOR are widely distributed throughout the animal kingdom and probably originated from a single ancestor gene coding for a dehydrogenase form of XOR. While a single mammalian XOR is known, various mammalian AOX isoenzymes have been identified. The complement of active mammalian AOX genes varies from one in humans (AOX1) to four in rodents (AOX1, AOX2, AOX3, and AOX4). In humans, the AOX1 and XOR genes are found on different arms of chromosome 2 and are both subject to complex regulation, the precise details of which have not yet been fully characterized. The 3D structures of bovine milk XOR, human AOX1, and mouse AOX3 are available and have provided valuable information on the catalytic mechanism of molybdenum hydroxylases. This chapter provides an overview of the current knowledge on the structure, evolution, and function of these highly complex enzymes.

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Regular ArticleIn Vitro Study of 6-mercaptopurine Oxidation Catalysed by Aldehyde Oxidase and Xanthine Oxidase

Summary:

Clin. Gastroenterol. Hepatol.

Biochem. Pharmacol.

Biochem. Pharmacol.

General Pharmacology

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Biochem. Pharmacol.

I. Purification and properties. J. Biol. Chem.

Anal. Biochem.

Inhibition studies with oxipurinol. Biochem. Pharmcol.

Biochem. Pharmacol.

Biochem. Pharmacol.

Clin. Chim. Acta.

The pharmacology and metabolism of the thiopurine drugs 6-mercaptopurine and azathioprine

Drug Metab. Rev.

Antineoplastic agents

Drug interactions of clinical significance. Drug Saf.

Regular Article
In Vitro Study of 6-mercaptopurine Oxidation Catalysed by Aldehyde Oxidase and Xanthine Oxidase