Discovery of Novel Reductive Elimination Pathway for 10-Hydroxywarfarin

Abstract

Coumadin (R/S-warfarin) anticoagulant therapy is highly efficacious in preventing the formation of blood clots; however, significant inter-individual variations in response risks over or under dosing resulting in adverse bleeding events or ineffective therapy, respectively. Levels of pharmacologically active forms of the drug and metabolites depend on a diversity of metabolic pathways. Cytochromes P450 play a major role in oxidizing R- and S-warfarin to 6-, 7-, 8-, 10-, and 4′-hydroxywarfarin, and warfarin alcohols form through a minor metabolic pathway involving reduction at the C11 position. We hypothesized that due to structural similarities with warfarin, hydroxywarfarins undergo reduction, possibly impacting their pharmacological activity and elimination. We modeled reduction reactions and carried out experimental steady-state reactions with human liver cytosol for conversion of rac-6-, 7-, 8-, 4′-hydroxywarfarin and 10-hydroxywarfarin isomers to the corresponding alcohols. The modeling correctly predicted the more efficient reduction of 10-hydroxywarfarin over warfarin but not the order of the remaining hydroxywarfarins. Experimental studies did not indicate any clear trends in the reduction for rac-hydroxywarfarins or 10-hydroxywarfarin into alcohol 1 and 2. The collective findings indicated the location of the hydroxyl group significantly impacted reduction selectivity among the hydroxywarfarins, as well as the specificity for the resulting metabolites. Based on studies with R- and S-7-hydroxywarfarin, we predicted that all hydroxywarfarin reductions are enantioselective toward R substrates and enantiospecific for S alcohol metabolites. CBR1 and to a lesser extent AKR1C3 reductases are responsible for those reactions. Due to the inefficiency of reactions, only reduction of 10-hydroxywarfarin is likely to be important in clearance of the metabolite. This pathway for 10-hydroxywarfarin may have clinical relevance as well given its anticoagulant activity and capacity to inhibit S-warfarin metabolism.

A physiologically‐based pharmacokinetic/pharmacodynamic modeling approach for drug–drug‐gene interaction evaluation of S‐warfarin with fluconazole

“…Metabolism of S‐warfarin and 6‐, 7‐, 10‐OH‐S‐warfarin is described using Michaelis–Menten kinetics. The Michaelis–Menten constant ( K m ) for CYP2C9, CYP3A4, UGT1A6, UGT1A1, and CBR1 is extracted from the literature 7,9,10,37 . The renal plasma clearance for S‐warfarin and metabolites was defined using first‐order kinetic and extracted from literature 14 .…”

“…The Michaelis–Menten constant ( K m ) for CYP2C9, CYP3A4, UGT1A6, UGT1A1, and CBR1 is extracted from the literature. 7 , 9 , 10 , 37 The renal plasma clearance for S‐warfarin and metabolites was defined using first‐order kinetic and extracted from literature. 14 OAT2‐mediated drug transport from the interstitial space to the intracellular in the liver is described using Michaelis–Menten equation too, the K m extracted from literature.…”

“…Further metabolism of 7‐ or 6‐OH‐S‐warfarin by UDP‐glucuronosyltransferases (UGTs), with UGT1A6 for 6‐OH‐S‐warfarin and UGT1A1 for 7‐OH‐S‐warfarin 7 . However, 10‐OH‐S‐warfarin is probably reduced by carbonyl reductase 1 (CBR1) 10 . Most studies suggested that S‐warfarin has three to five times higher anticoagulant activity than R‐warfarin 11–13 .…”

“… 7 However, 10‐OH‐S‐warfarin is probably reduced by carbonyl reductase 1 (CBR1). 10 Most studies suggested that S‐warfarin has three to five times higher anticoagulant activity than R‐warfarin. 11 , 12 , 13 In this study, we mainly consider the modeling of S‐warfarin.…”