The metabolism of [(14)C]-diclofenac in mice was investigated following a single oral dose of 10 mg/kg. The majority of the drug-related material was excreted in the urine within 24 h of administration (49.7 %). Liquid chromatographic analyses of urine and faecal extracts revealed extensive metabolism to at least 37 components, with little unchanged diclofenac excreted. Metabolites were identified using a hybrid linear ion-trap mass spectrometer via exact mass determinations of molecular ions and subsequent multi-stage fragmentation. The major routes of metabolism identified included: 1) conjugation with taurine; and 2) hydroxylation (probably at the 4'-and 5-arene positions) followed by conjugation to taurine, glucuronic acid or glucose. Ether, rather than acyl glucuronidation, predominated. There was no evidence for p-benzoquinone-imine formation (i.e. no glutathione or mercapturic acid conjugates were detected). A myriad of novel minor drug-related metabolites were also detected, including ribose, glucose, sulfate and glucuronide ether-linked conjugates of hydroxylated diclofenac derivatives. Combinations of these hydroxylated derivatives with acyl conjugates (glucose, glucuronide and taurine) or N-linked sulfation or glucosidation were also observed. Acyl- or amide-linked-conjugates of benzoic acid metabolites and several indolinone derivatives with further hydroxylated and conjugated moieties were also evident. The mechanisms involved in the generation of benzoic acid and indolinone products indicate the formation reactive intermediates in vivo that may possibly contribute to hepatotoxicity.

1. The distribution, metabolism, excretion and hepatic effects of fenclozic acid were investigated following a single oral dose of 10 mg/kg to hepatic reductase null (HRN) mice. 2. The majority of the [(14)C]-fenclozic acid was eliminated via the urine/aqueous cage wash, (55%) with a smaller portion excreted in the faeces, (5%). The total recovery of radioactivity in the excreta over the 72 h period studied was ca. 60%. 3. Metabolism of fenclozic acid in the HRN mice was entirely to the carboxylic acid function and was dominated by amino acid conjugation to glycine and taurine, with lesser amounts of an acyl glucuronide. 4. Whole body autoradiography of mice showed general distribution into all tissues except the brain. Radioactivity was still detectable in the kidney and liver of the HRN mice at 72 h post-dose. Covalent binding studies showed evidence of binding to kidney, liver and plasma proteins however, the degree of binding was less than 50 pmol equiv/mg protein for all tissues. 5. The HRN mouse appears to be a useful in vivo model for the study of the Phase II conjugation metabolism of fenclozic acid in the absence of hepatic cytochrome P450-related oxidative metabolism.

The distribution, metabolism, excretion and hepatic effects of the human hepatotoxin fenclozic acid were investigated following single oral doses of 10 mg/kg to normal and bile duct-cannulated male C57BL/6J mice. Whole body autoradiography showed distribution into all tissues except the brain, with radioactivity still detectable in blood, kidney and liver at 72 h post-dose. Mice dosed with [14C]-fenclozic acid showed acute centrilobular hepatocellular necrosis, but no other regions of the liver were affected. The majority of the [14C]-fenclozic acid-related material recovered was found in the urine/aqueous cage wash, (49%) whilst a smaller portion (13%) was eliminated via the faeces. Metabolic profiles for urine, bile and faecal extracts, obtained using liquid chromatography and a combination of mass spectrometric and radioactivity detection, revealed extensive metabolism of fenclozic acid in mice that involved biotransformations via both oxidation and conjugation. These profiling studies also revealed the presence of glutathione-derived metabolites providing evidence for the production of reactive species by mice administered fenclozic acid. Covalent binding to proteins from liver, kidney and plasma was also demonstrated, although this binding was relatively low (less than 50 pmol eq./mg protein).Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-016-1894-5) contains supplementary material, which is available to authorized users.

The distribution, metabolism, excretion and hepatic effects of diclofenac were investigated following a single oral dose of 10 mg/kg to wild type and hepatic reductase null (HRN) mice. For the HRN strain the bulk of the [(14)C]-diclofenac-related material was excreted in the urine/aqueous cagewash within 12 h of administration (~82%) with only small amounts eliminated via the faeces (~2% in 24 h). Wild type mice excreted the radiolabel more slowly with ca. 52 and 15% of the dose recovered excreted in urine and faeces, respectively, by 24 h post dose. The metabolic profiles of the HRN mice were dominated by acyl conjugation to either taurine or glucuronic acid. Wild type mice produced relatively small amounts of the acyl glucuronide. Whole Body Autoradiography (WBA) of mice sacrificed at 24 h post dose indicated increased retention of radioactivity in the livers of HRN mice compared to wild type mice. Covalent binding studies showed no differences between the two strains. Metabolism of diclofenac in HRN mice involved mainly acyl glucuronide formation and taurine amide conjugation. This mouse model may find utility in understanding the impact of reactive metabolite formation via routes that involve the production of acyl-CoA or acyl glucuronides of acidic drugs.

Adenosine is generated continuously in cultured HUVEC under normal conditions, but is immediately recycled via adenosine kinase. In ATP depleted cells, nucleotide catabolism proceeds predominantly via intracellular dephosphorylation of AMP with a small contribution from the extracellular dephosphorylation pathway. The capacity of the AMP deamination pathway in endothelium is small and the flux through xanthine oxidoreductase is minimal.