title: Carbonyl reduction of warfarin: Identification and characterization of human warfarin reductases.
abstract: Warfarin is a widely used anticoagulant and, unfortunately, is a drug that is commonly implicated in serious adverse events including fatalities. Although several factors, including the metabolism of warfarin via CYP450, have been reported to affect the safety and efficacy of warfarin therapy, the wide variance in the warfarin dosage in patients has not been completely clarified. In addition to the oxidative metabolism of warfarin mediated by CYP450, reductive metabolism is involved in warfarin biotransformation. However, the reductive metabolism of warfarin has been largely unexplored and deserves further investigation. We studied warfarin reduction by human liver fractions and found a 9-fold higher velocity of warfarin reduction in the cytosol than in microsomes (Vmax=77.2 vs. 8.7pmol/mgprotein/min, respectively). Furthermore, of nine recombinant cytosolic carbonyl reducing enzymes tested for their ability to reduce warfarin, AKR1C3 and CBR1 were identified as warfarin reductases and their kinetic parameters were determined. The internal clearance of warfarin was 3 orders of magnitude higher with AKR1C3 than with CBR1 (CLint=65.922 vs. 0.070μl/mgprotein/min, respectively). This is the first time that warfarin reducing enzymes in human liver subcellular fraction have been identified. Moreover, we have described the chiral aspects of warfarin reduction using an HPLC method that enabled the detection of individual warfarin alcohol stereoisomers. Cytosol and AKR1C3 exhibit the stereoselective metabolism of (R)-warfarin to preferentially form (SR)-warfarin alcohol as the primary in vivo metabolite of warfarin. On the other hand, microsomes and CBR1 preferentially reduce (S)-warfarin to form (RS)-warfarin alcohol and (SS)-warfarin alcohol, respectively.
authors.affiliation: Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Heyrovského 1203, CZ-50005 Hradec Králové, Czech Republic. Electronic address: Petra.Malatkova@faf.cuni.cz.
authors.forename: Petra
authors.initials: P
authors.lastname: Malátková
authors.affiliation: Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Heyrovského 1203, CZ-50005 Hradec Králové, Czech Republic. Electronic address: simonka.s@centrum.sk.
authors.forename: Simona
authors.initials: S
authors.lastname: Sokolová
authors.affiliation: Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovského 1203, CZ-50005 Hradec Králové, Czech Republic. Electronic address: havlikova@faf.cuni.cz.
authors.forename: Lucie
authors.initials: L
authors.lastname: Chocholoušová Havlíková
authors.affiliation: Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Heyrovského 1203, CZ-50005 Hradec Králové, Czech Republic. Electronic address: vladimir.wsol@faf.cuni.cz.
authors.forename: Vladimír
authors.initials: V
authors.lastname: Wsól
mesh_terms: D015096:3-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000449:Aldehyde Reductase / Q000378:metabolism; D000074425:Aldo-Keto Reductase Family 1 Member C3; D000925:Anticoagulants / Q000378:metabolism*; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D006911:Hydroxyprostaglandin Dehydrogenases / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000378:metabolism; D013237:Stereoisomerism; D014859:Warfarin / Q000378:metabolism*
chemical_list: D000925:Anticoagulants; D003470:Culture Media; D007527:Isoenzymes; D011994:Recombinant Proteins; D014859:Warfarin; D015096:3-Hydroxysteroid Dehydrogenases; D000429:Alcohol Oxidoreductases; D006911:Hydroxyprostaglandin Dehydrogenases; C531129:CBR1 protein, human; C580929:AKR1A1 protein, human; C484670:AKR1B1 protein, human; D000449:Aldehyde Reductase; C487341:AKR1C3 protein, human; D000074425:Aldo-Keto Reductase Family 1 Member C3
keywords: (RS)-warfarin alcohol (PubChem CID: 54736486); (SS)-warfarin alcohol (PubChem CID: 56842683); Aldo–keto reductase (AKR); Carbonyl reducing enzymes; Carbonyl reductase (CBR); Drug metabolism; Short-chain dehydrogenase/reductase; Warfarin; Warfarin (PubChem CID: 54678486); Warfarin alcohol (PubChem CID: 54712717)