5-fluoro-2-pyrimidinone, a liver aldehyde oxidase-activated prodrug of 5-fluorouracil

doi:10.1016/0006-2952(95)98508-7

Biochemical Pharmacology

Volume 49, Issue 8, 18 April 1995, Pages 1111-1116

https://doi.org/10.1016/0006-2952(95)98508-7 Get rights and content

Abstract

5-Fluorouracil (5-FU) is an effective antitumor agent used in treating various cancers. Because of its metabolism by intestinal and other cells, 5-FU has an inconsistent bioavailability that limits its oral use. 5-Fluoro-2-pyrimidinone (5-FP), a 5-FU prodrug, was synthesized and found to be converted to 5-FU by aldehyde oxidase, an enzyme present in high concentrations in the livers of mice and humans but not in the gastrointestinal tract. Using BDF1 mice, the pharmacokinetics of 5-FP were studied and compared with those of 5-FU. The bioavailability of 5-FP given orally was 100% at a dosage of 25 mg/kg and 78% at a dosage of 50 mg/kg. The half-lives of both doses of 5-FP were at least 2-fold longer than the half-lives of the same doses of 5-FU, and the clearance rates of 5-FP were 3-fold slower. 5-FP was converted rapidly to 5-FU in vivo. The resulting 5-FU was measured at a steady-state level of 40–70 μM in plasma, at a dosage of 25 mg/kg, that was sustained for at least 4 hr. Also, when given orally, 5-FP was shown to have potent activity against Colon 38 tumor cells and P388 leukemia cells in mice. The therapeutic index of 5-FP was similar to that of 5-FU in these mouse tumor models. The potential clinical use of 5-FP as a prodrug of 5-FU should be considered.

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Potential interactions of new drugs with AOX and consequences for therapeutic efficacy, half-life and availability. AOX1 can metabolically activate some prodrugs, which makes the enzyme an interesting pharmacological target for chemotherapy [51–53]. Metabolic activation of prodrugs by AOX1 has already been exploited to overcome pharmacokinetic problems and to improve bioavailability, as exemplified by the clinical development of the 5-fluoruracil precursor, 5-fluoro-2-pyrimidinone, and the 5-iodo-2-deoxyuridine precursor, 5-iodo-2-pyrimidinone-2-deoxyribose [54–58].
The aldehyde oxidases (AOXs) are a small sub-family of cytosolic molybdo-flavoenzymes, which are structurally conserved proteins and broadly distributed from plants to animals. AOXs play multiple roles in both physiological and pathological processes and AOX inhibition is of increasing significance in the development of novel drugs and therapeutic strategies. This review provides an overview of the evolution and the action mechanism of AOX and the role of each domain. The review provides an update of the polymorphisms in the human AOX. This review also summarises the physiology of AOX in different organs and its role in drug metabolism. The inhibition of AOX is a promising therapeutic treatment for cancer, obesity, aging and amyotrophic lateral sclerosis.
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Similarly, 5-FP was activated to 5-FU very efficiently by AO (KM value of 220 μM and Vmax of 8 nmol/min/mg). However, the prodrug approach failed due to lack of selectivity and or similar cytostatic activity as 5-FU (dosed by itself) (Guo et al., 1995; Porter et al., 1994). Despite these efforts, exploitation of AO for prodrug activation has not gained traction.
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.
Xanthine Oxidoreductase and Aldehyde Oxidases
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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.
Xanthine Oxidoreductase and Aldehyde Oxidase
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Aldehyde oxidase (AO) 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) (FAD) 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 dicucleotide – oxidized form). Reduction of oxygen can generate reactive oxygen species (ROS), which have been implicated in a variety of protective and pathophysiological functions. AO and XOR are widely distributed throughout the animal kingdom and probably originated from a single ancestor gene coding for a dehydrogenase form of XOR. In humans, AO 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. Although the 3D structures of human native XOR or AO are not available, elucidation of the crystal structures of bovine milk XOR and bacterial aldehyde oxidoreductases (AORs) has provided valuable information on the catalytic mechanism of the molybdenum hydroxylases. This chapter provides an overview of the current knowledge on these highly complex enzymes.
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To aid in the clinical evaluation of zebularine, a potential oral antitumor agent, we initiated studies on the metabolism of zebularine in liver cytosol from humans and other mammals. Metabolism by aldehyde oxidase (AO, EC 1.2.3.1) was the major catabolic route, yielding uridine as the primary metabolite, which was metabolized further to uracil by uridine phosphorylase. The inhibition of zebularine metabolism was studied using raloxifene, a known potent inhibitor of AO, and 5-benzylacyclouridine (BAU), a previously undescribed inhibitor of AO. The Michaelis–Menten kinetics of aldehyde oxidase and its inhibition by raloxifene and BAU were highly variable between species.

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Research paper5-fluoro-2-pyrimidinone, a liver aldehyde oxidase-activated prodrug of 5-fluorouracil

Abstract

Am J Surg

Pharmacol Ther

Biochem Pharmacol

Biochem Pharmacol

Gastroenterology

The natural history of primary and secondary malignant tumors of the liver. I. The prognosis of patients with hepatic metastases from colonic and rectal carcinoma by laparotomy

Cancer

Phase I trial of hepatic artery infusion of 5-iodo-2′-deoxyuridine and 5-fluorouracil in patients with advanced hepatic malignancy: Biochemically based combination chemotherapy

Cancer Res

Fluorinated pyrimidines, a new class of tumor-inhibitory compounds

Nature

The pharmacology of the fluoropyrimidines

Pharmacol Rev

Purine and pyrimidine antimetabolites

Pyrimidine antagonists

A clinicalpharmacological evaluation of hepatic arterial infusions of 5-fluoro-2′-deoxyuridine and 5-fluorouracil

Cancer Res

Fluorouracil therapy in patients with carcinoma of the large bowel: A pharmacokinetic comparison of various rates and routes of administration

Clin Pharmacokinet

Research paper
5-fluoro-2-pyrimidinone, a liver aldehyde oxidase-activated prodrug of 5-fluorouracil