Warfarin metabolites: Stereochemical aspects of protein binding and displacement by phenylbutazone

Chan, Eli; McLachlan, Andrew J.; Rowland, Malcolm

doi:10.1002/chir.530050808

Cited by 13 publications

(12 citation statements)

References 20 publications

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“…Although the binding affinity of hydroxywarfarins for CYP2C9 is constant, the potential for in vivo inhibition of CYP2C9 is augmented by the differing plasma protein binding properties of warfarin and its metabolites. S -Warfarin has high affinity for plasma proteins (~ 99% bound) (16), while 6-, 7-, and 8-hydroxywarfarin bind plasma proteins with 3- to 4-fold lower affinity (17). 4′-Hydroxywarfarin is thought to bind human plasma albumin with affinity comparable to that of warfarin (16), but the affinity of 10-hydroxywarfarin is unknown.…”

Section: Discussionmentioning

confidence: 99%

“…We therefore estimate the mean level of unbound S -warfarin in human plasma to be 2.7 ng/mL (8). We estimate the mean unbound concentration of 7- and 10-hydroxywarfarin to be 6.2 and 5.8 ng/mL, respectively, given that ~4% of the hydroxywarfarins are unbound (17). We expect the inhibitory effect of each of these metabolites to be additive; therefore, 7-, and 10-hydroxywarfarin have a combined concentration approximately 4.5 times higher than that of S -warfarin.…”

Section: Discussionmentioning

confidence: 99%

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Hydroxywarfarin Metabolites Potently Inhibit CYP2C9 Metabolism of S-Warfarin

Jones

Kim

Guderyon

et al. 2010

Chem. Res. Toxicol.

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Coumadin (R/S-warfarin) anticoagulant therapy poses a risk to over 50 million Americans, in part due to interpersonal variation in drug metabolism. Consequently, it is important to understand how metabolic capacity is influenced among patients. Cytochrome P450s (P450 or CYP for a specific isoform) catalyze the first major step in warfarin metabolism to generate five hydroxywarfarins for each drug enantiomer. These primary metabolites are thought to reach at least 5-fold higher levels in plasma than warfarin. We hypothesized that hydroxywarfarins inhibit the hydroxylation of warfarin by CYP2C9, thereby limiting enzymatic capacity toward S-warfarin. To test this hypothesis, we investigated the ability of all five racemic hydroxywarfarins to block CYP2C9 activity toward S-warfarin using recombinant enzyme and human liver microsomes. We initially screened for the inhibition of CYP2C9 by hydroxywarfarins using a P450-Glo assay to determine IC50 values for each hydroxywarfarin. Compared to the substrate, CYP2C9 bound its hydroxywarfarin products with less affinity but retained high affinity for 10- and 4′-hydroxywarfarins, products from CYP3A4 reactions. S-Warfarin steady-state inhibition studies with recombinant CYP2C9 and pooled human liver microsomes confirmed that hydroxywarfarin products from CYP reactions possess the capacity to competitively inhibit CYP2C9 with biologically relevant inhibition constants. Inhibition of CYP2C9 by 7-hydroxywarfarin may be significant given its abundance in human plasma, despite its weak affinity for the enzyme. 10-Hydroxywarfarin, which has been reported as the second most abundant plasma metabolite, was the most potent inhibitor of CYP2C9, displaying approximately 3-fold higher affinity than S-warfarin. These results indicate that hydroxywarfarin metabolites produced by CYP2C9 and other CYPs may limit metabolic capacity toward S-warfarin through competitive inhibition. Subsequent processing of hydroxywarfarins to secondary metabolites, such as hydroxywarfarin glucuronides, could suppress product feedback inhibition, and therefore could play an important role in the modulation of metabolic pathways governing warfarin inactivation and elimination.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Hydroxywarfarin Metabolites Potently Inhibit CYP2C9 Metabolism of S-Warfarin

Jones

Kim

Guderyon

et al. 2010

Chem. Res. Toxicol.

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“…The injector loop was loaded with a 1.5 mL portion of the sample solution and connected to the mobile phase flow (1.0 mL/min) for 60 s. Therefore, the actual injection volume was 1.0 mL. 19,32,[36][37][38][39][40] The effect of Ph on the binding between Wf and HSA is enantioselective. The injection volume shown in Figures 3-5 (except for 20 µL) was also the actual sample volume introduced by the "injectorreswitching technique" with appropriate loading volume and reswitching time.…”

Section: Resultsmentioning

confidence: 99%

“…32,40 Two possible hypotheses can be considered to explain the change in the enantioselectivity by addition of Ph, as pointed out by Chan et al 40 One explanation is that Ph induces a conformational change in the Wf binding site on the albumin molecule which produces a differential change in the affinities with Wf enantiomers. 32,40 Two possible hypotheses can be considered to explain the change in the enantioselectivity by addition of Ph, as pointed out by Chan et al 40 One explanation is that Ph induces a conformational change in the Wf binding site on the albumin molecule which produces a differential change in the affinities with Wf enantiomers.…”

Section: Resultsmentioning

confidence: 99%

Protein-Binding High-Performance Frontal Analysis of (R)- and (S)-warfarin on HSA with and without Phenylbutazone

Shibukawa

Tokunaga

et al. 1997

Journal of Pharmaceutical Sciences

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Applicability of high-performance frontal analysis (HPFA) to the stereoselective study of drug-drug interaction upon plasma protein binding has been investigated. Racemic warfarin and phenylbutazone were used as model drugs. An on-line HPFA/HPLC system consisting of a HPFA column (diol-silica column), an extraction column, and a chiral separation column was developed, and human serum albumin solution containing racemic warfarin and/or phenylbutazone was injected directly to the HPFA column. When the injection volume was large enough, the binding equilibrium in the sample solution was reproduced in the column, and consequently a plateau region appeared on the chromatogram. This plateau region contains unbound drug(s). A given volume of eluent in the plateau part was transferred into the extraction column by column-switching. The concentrated drug(s) was then transferred to the chiral separation column to determine the unbound concentrations of the enantiomers and/or the competitor. The results agreed with those obtained by a conventional ultrafiltration-HPLC method. The influence of phenylbutazone upon the protein binding of warfarin is enantioselective. In warfarin and human serum albumin mixed solution, the unbound concentration of (R)-warfarin was 1.22 times higher than that of the S-isomer. By addition of phenylbutazone, the unbound concentration of (S)-warfarin increased more than that of (R)-warfarin, resulting in the reversed enantioselectivity, i.e., the unbound concentration of (S)-warfarin became 1.19 times larger than that of (R)-warfarin. The present method was also applicable to human plasma samples.

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“…An important driver in this process is the concentration or plasma level of 7-hydroxywarfarin in warfarin patients, which have been reported to range from 6 to 50 nM for R -7-hydroxywarfarin and 188 to 1140 nM for S -7-hydroxywarfarin [5, 7, 39]. The amount accessible for glucuronidation is actually 20-fold lower than these reported ranges, because only 4.49 % of R -7-hydroxywarfarin and 4.27 % of S -7-hydroxywarfarin are not bound to plasma proteins [40]. Consequently, plasma levels of 7-hydoxywarfarin available for glucuronidation are sub-nanomolar for R -7-hydroxywarfarin and mid to low nanomolar for S-7-hydroxywarfarin.…”

Section: Discussionmentioning

confidence: 99%

Multiple UDP-glucuronosyltransferases in human liver microsomes glucuronidate both R- and S-7-hydroxywarfarin into two metabolites

Pugh

Pouncey

Hartman

et al. 2014

Archives of Biochemistry and Biophysics

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The widely used anticoagulant Coumadin (R/S-warfarin) undergoes oxidation by cytochromes P450 into hydroxywarfarins that subsequently become conjugated for excretion in urine. Hydroxywarfarins may modulate warfarin metabolism transcriptionally or through direct inhibition of cytochromes P450 and thus, UGT action toward hydroxywarfarin elimination may impact levels of the parent drugs and patient responses. Nevertheless, relatively little is known about conjugation by UDP-glucuronosyltransferases in warfarin metabolism. Herein, we identified probable conjugation sites, kinetic mechanisms and hepatic UGT isoforms involved in microsomal glucuronidation of R- and S-7-hydroxywarfarin. Both compounds underwent glucuronidation at C4 and C7 hydroxyl groups based on elution properties and spectral characteristics. Their formation demonstrated regio- and enantioselectivity by UGTs and resulted in either Michaelis-Menten or substrate inhibition kinetics. Glucuronidation at the C7 hydroxyl group occurred more readily than at the C4 group, and the reaction was overall more efficient for R-7-hydroxywarfarin due to higher affinity and rates of turnover. The use of these mechanisms and parameters to model in vivo clearance demonstrated that contributions of substrate inhibition would lead to underestimation of metabolic clearance than that predicted by Michaelis-Menten kinetics. Lastly, these processes were driven by multiple UGTs indicating redundancy in glucuronidation pathways and ultimately metabolic clearance of R- and S-7-hydroxywarfarin.

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Warfarin metabolites: Stereochemical aspects of protein binding and displacement by phenylbutazone

Cited by 13 publications

References 20 publications

Hydroxywarfarin Metabolites Potently Inhibit CYP2C9 Metabolism of S-Warfarin

Hydroxywarfarin Metabolites Potently Inhibit CYP2C9 Metabolism of S-Warfarin

Protein-Binding High-Performance Frontal Analysis of (R)- and (S)-warfarin on HSA with and without Phenylbutazone

Multiple UDP-glucuronosyltransferases in human liver microsomes glucuronidate both R- and S-7-hydroxywarfarin into two metabolites

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