Coumadin (R-, S-warfarin)is a challenging drug to accurately dose, both initially and for maintenance, because of its narrow therapeutic range and wide interpatient variability and is typically administered as a racemic (Rac) mixture, which complicates the biotransformation pathways. The goal of the current work was to identify the human UDP-glucuronosyltransferases (UGTs) involved in the glucuronidation of the separated R-and S-enantiomers of 6-, 7-, and 8-hydroxywarfarin and the possible interactions between these enantiomers. The kinetic and inhibition constants for human recombinant 1A family UGTs toward these separated enantiomers have been assessed using high-performance liquid chromatography (HPLC)-UV-visible analysis, and product confirmations have been made using HPLC-mass spectrometry/mass spectrometry. We found that separated R-and S-enantiomers of 6-, 7-, and 8-hydroxywarfarin demonstrate significantly different glucuronidation kinetics and can be mutually inhibitory. In some cases significant substrate inhibition was observed, as shown by K m , V max , and K i , comparisons. In particular, UGT1A1 and extrahepatic UGT1A10 have significantly higher capacities than other isoforms for S-7-hydroxywarfarin and R-7-hydroxywarfarin glucuronidation, respectively. Activity data generated using a set of well characterized human liver microsomes supported the recombinant enzyme data, suggesting an important (although not exclusive) role for UGT1A1 in glucuronidation of the main warfarin metabolites, including Rac-6-and 7-hydroxywarfarin and their R-and S-enantiomers in the liver. This is the first demonstration that the R-and S-enantiomers of hydroxywarfarins are glucuronidated, with significantly different enzymatic affinity and capacity, and supports the importance of UGT1A1 as the major hepatic isoform involved.

Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme, synthesized in the liver and secreted into the blood. PON1 displays antioxidant properties and is involved in organophosphorous compounds and oxidized lipids degradation. Because of these beneficial effects, pharmacological regulation of PON1 appears to be highly relevant in toxicology and cardiology. Recent studies undertaken on the regulation of the PON1 promoter in our laboratory have identified resveratrol, through its activation of the Aryl hydrocarbon Receptor (AhR), as a putative inducer of PON1. We have tested a new modulator of AhR, (Z)-2,3-bis (4-nitrophenyl)-acrylonitrile, and established that it is a more potent inducer of PON1 at the mRNA, protein and enzymatic activity as compared to resveratrol. It also acts by activating the AhR. However, in contrast with traditional AhR agonists, it does not induce cyp1A1 transcription. (Z)-2,3-bis (4-nitrophenyl)-acrylonitrile is therefore a specific AhR modulator targeting PON1.