Warfarin Enantiomers Research Articles

Potentiation of anticoagulant effect of warfarin caused by enantioselective metabolic inhibition by the uricosuric agent benzbromarone.

To clarify the mechanism(s) for the interaction between warfarin and benzbromarone, a uricosuric agent, and to predict changes in the in vivo pharmacokinetics of (S)-warfarin from in vitro data. Warfarin enantiomers and benzbromarone in serum, 7-hydroxywarfarin in urine, and serum unbound fractions of warfarin enantiomers were measured in patients with heart disease given warfarin with (n = 13) or without (n = 18) oral benzbromarone (50 mg/d). In vitro inhibition constants (K(i)) of benzbromarone for (S)-warfarin 7-hydroxylation were determined with use of human CYP2C9 and liver microsomes. The magnitude of changes in the formation clearance for 7-hydroxylation (CLf), the unbound oral clearance (CL(oral,u)), and the oral clearance (CL(oral)) for (S)-warfarin were predicted by equations incorporating the in vitro Ki, the theoretical maximum unbound hepatic benzbromarone concentration, and the fractions of warfarin eliminated through metabolism and of CYP2C9-mediated metabolic reaction susceptible to inhibition by benzbromarone. The patients given warfarin with benzbromarone required a 36% less (P < .01) warfarin dose than those given warfarin alone (2.5 versus 3.9 mg/d) to attain similar international normalized ratios (2.1 and 2.2, respectively), and the former had 65%, 53%, and 54% lower (P < .05 or P < .01) CLf, CL(oral),u, and CL(oral) for (S)-warfarin than the latter, respectively. In contrast, no significant differences were observed for (R)-warfarin kinetics between the groups. Benzbromarone was found to be a potent competitive inhibitor (Ki < 0.01 micromol/L) for (S)-warfarin 7-hydroxylation mediated by CYP2C9. The average changes in the in vivo CLf, CL(oral),u, and CL(oral)values for (S)-warfarin induced by benzbromarone were largely predictable by the proposed equations. Benzbromarone would intensify anticoagulant response of warfarin through an enantioselective inhibition of CYP2C9-mediated metabolism of pharmacologically more potent (S)-warfarin. The magnitude of changes in the in vivo warfarin kinetics may be predicted by in vitro data.

Effect of Montelukast on the Pharmacokinetics and Pharmacodynamics of Warfarin in Healthy Volunteers

Montelukast, a cysteinyl leukotriene receptor antagonist, is being developed for the treatment of asthma and related diseases. This study was designed to evaluate whether montelukast at clinically used dosage levels would interfere with the anticoagulant effect of warfarin. In a two-period, double-blind, randomized crossover study, 12 healthy male subjects received a single oral dose of 30 mg warfarin on the 7th day of a 12-day treatment with montelukast, 10 mg daily by mouth, or a placebo. Montelukast had no significant effect on the area under the plasma concentration-time curves and peak plasma concentrations of either R- or S-warfarin. However, slight but statistically significant decreases in time to peak concentration of both warfarin enantiomers and in elimination half-life of the less potent R-warfarin were observed in the presence of montelukast. These changes were not considered as clinically relevant. Montelukast had no significant effect on the anticoagulant effect of warfarin, as assessed by the international normalized ratio (INR) for prothrombin time (AUC0-144 and INR maximum). The results of this study suggest that a clinically important interaction between these drugs is unlikely to occur in patients requiring concomitant administration of both drugs.

Enzymatic determinants of the substrate specificity of CYP2C9: role of B'-C loop residues in providing the pi-stacking anchor site for warfarin binding.

Previous modeling efforts have suggested that coumarin ligand binding to CYP2C9 is dictated by electrostatic and pi-stacking interactions with complementary amino acids of the protein. In this study, analysis of a combined CoMFA-homology model for the enzyme identified F110 and F114 as potential hydrophobic, aromatic active-site residues which could pi-stack with the nonmetabolized C-9 phenyl ring of the warfarin enantiomers. To test this hypothesis, we introduced mutations at key residues located in the putative loop region between the B' and C helices of CYP2C9. The F110L, F110Y, V113L, and F114L mutants, but not the F114Y mutant, expressed readily, and the purified proteins were each active in the metabolism of lauric acid. The V113L mutant metabolized neither (R)- nor (S)-warfarin, and the F114L mutant alone displayed altered metabolite profiles for the warfarin enantiomers. Therefore, the effect of the F110L and F114L mutants on the interaction of CYP2C9 with several of its substrates as well as the potent inhibitor sulfaphenazole was chosen for examination in further detail. For each substrate examined, the F110L mutant exhibited modest changes in its kinetic parameters and product profiles. However, the F114L mutant altered the metabolite ratios for the warfarin enantiomers such that significant metabolism occurred for the first time on the putative C-9 phenyl anchor, at the 4'-position of (R)- and (S)-warfarin. In addition, the Vmax for (S)-warfarin 7-hydroxylation decreased 4-fold and the Km was increased 13-fold by the F114L mutation, whereas kinetic parameters for lauric acid metabolism, a substrate which cannot interact with the enzyme by a pi-stacking mechanism, were not markedly affected by this mutation. Finally, the F114L mutant effected a greater than 100-fold increase in the Ki for inhibition of CYP2C9 activity by sulfaphenazole. These data support a role for B'-C helix loop residues F114 and V113 in the hydrophobic binding of warfarin to CYP2C9, and are consistent with pi-stacking to F114 for certain aromatic ligands.

Site I on human albumin: differences in the binding of (R)- and (S)-warfarin.

The binding of drugs known to interact with area I on human serum albumin (HSA) was investigated using a chiral stationary phase obtained by anchoring HSA to a silica matrix. In particular, this high-pressure affinity chromatography selector was employed to study the binding properties of the individual enantiomers of warfarin. The pH and composition of the mobile phase modulate the enantioselective binding of warfarin. Displacement chromatography experiments evidenced significant differences in the binding of the warfarin enantiomers to site I. The (S)-enantiomer was shown to be a direct competitor for (R)-warfarin, while (R)-warfarin was an indirect competitor for the (S)-enantiomer. Salicylate directly competed with (R)-warfarin and indirectly with (S)-warfarin. This behavior was confirmed by difference CD experiments, carried out with the same [HSA]/[drug] system in solution.

Stereoselective Binding of Warfarin and Ketoprofen to Human Serum Albumin Determined by Microdialysis Combined with HPLC

Microdialysis sampling technique combined with high performance liquid chromatography (HPLC) was utilized to determine the stereoselective binding of warfarin and ketoprofen to human serum albumin (HSA) in solution. The HPLC conditions for separation of warfarin and ketoprofen enantiomers on HSA column were optimized. The unbound enantiomers in the mixed buffer (0.067 M phosphate and pH 7.4) solution of the drug racemate with HAS were sampled with microdialysis probe, and the amount of them was determined by HPLC on HSA column. It was observed that the unbound concentrations of R-warfarin and R-ketoprofen are about 1.08∼1.34-and 1.08∼1.15-fold of S-warfarin and S-ketoprofen, respectively, which indicated that the S-enantiomers bind to HSA more strongly than R-enantiomers to HSA. The interaction parameters for binding of warfarin and ketoprofen enantiomers to HSA including binding constants and number of binding site were obtained by the Scatchard analysis of experimental data, and the stereoselectivity (α) calculated by the binding constants of warfarin enantiomers to HSA is 1.92, but that of ketoprofen enantiomers is very close. HSA exhibits stronger stereoselectivity to warfarin racemate than to ketoprofen racemate.

Chiral phase analysis of warfarin enantiomers in patient plasma in relation to CYP2C9 genotype

A direct chiral-phase high-performance liquid chromatographic method for measuring the ratio of S-warfarin/ R-warfarin in patient plasma is described. Plasma samples are first extracted using solid-phase C 18 extraction columns, and the concentrated extracts analyzed using an ( R, R) Whelk-O 1 column with a mobile phase of 0.5% glacial acetic acid in acetonitrile. The resulting chromatography provides baseline resolution of the warfarin enantiomers and internal standard (racemic ethylwarfarin), and is free from interference from other plasma components. Calibration curves were linear (mean r 2 of 0.999 for both enantiomers) over the concentration range 0.25–1.5 μg/ml. The intra-day and inter-day coefficients of variation for analysis of plasma spiked with 0.33 μg/ml S-warfarin and 0.67 μg/ml R-warfarin ( S/ R=0.5:1) was less than 7% for each enantiomer, with an accuracy of more than 93%. Plasma extracts from thirty-one patients homozygous for wild-type CYP2C9*1 provided an S/ R ratio of 0.51±0.15. Two warfarin patients homozygous for the mutant CYP2C9*2 and CYP2C9*3 alleles exhibited elevated S/ R ratios relative to the mean for individuals homozygous for the wild-type CYP2C9*1 allele. This method is suitable for population studies aimed at establishing the effect of polymorphic expression of CYP2C9 alleles on S-warfarin elimination in humans.

新しい血清遊離形ワルファリン光学異性体濃度測定法による同薬の血清濃度・抗凝固効果関係の検討

新しい血清遊離形ワルファリン光学異性体濃度測定法による同薬の血清濃度・抗凝固効果関係の検討

Determination of unbound warfarin enantiomers in human plasma and 7-hydroxywarfarin in human urine by chiral stationary-phase liquid chromatography with ultraviolet or fluorescence and on-line circular dichroism detection

Enantiomers of warfarin and 7-hydroxywarfarin in human plasma and urine, respectively, were determined by high-performance liquid chromatography using a cellulose-derivative column with UV or fluorescent detection, and their absolute configuration was determined simultaneously by a circular dichroism spectropolarimeter connected in series. Enantiomers of warfarin and its major metabolites [i.e., ( R)-6-hydroxywarfarin, ( S)-7-hydroxywarfarin and ( RS)-warfarin alcohol] were well resolved. The method was precise and sensitive: within- and between-day coefficients of variation were <9.6% for warfarin enantiomers in plasma and <7.1% for 7-hydroxywarfarin enantiomers in urine, respectively, and the lower detection limits were 20 ng/ml for ( R)-warfarin, 40 ng/ml for ( S)-warfarin, 2.5 ng/ml ( R)-7-hydroxywarfin and 4.5 ng/ml for( S)-7-hydroxywarfarin in 0.5 ml of both plasma and urine. The ultrafiltration technique was used for determining unbound concentrations of warfarin enantiomers in plasma using [ 14C]warfarin enantiomers resolved by the present HPLC system. Clinical applicability of the method was evaluated by determining unbound concentrations of warfarin enantiomers in five consecutive plasma samples obtained from a patient exhibiting an unstable anticoagulant response to warfarin (4 mg/day, p.o.). Results indicated that the present method would be useful in clarifying factors responsible for a large intra- and inter-patient variability in warfarin effects with regard to unbound plasmaenantiomer pharmacokinetics.

THE CHARACTERIZATION OF POTENT NOVEL WARFARIN ANALOGS

Racemic sodium warfarin, Coumadin,® is widely used in the prevention of thromboembolic disease. The present study was undertaken to characterize three novel classes of warfarin analogs, and to compare them with the warfarin enantiomers. All three classes of compounds inhibit vitamin K epoxide reductase, the enzyme inhibited by racemic warfarin. The alcohol and the ester analogs have reduced protein binding compared with R-(+)-warfarin 1 1 R-(+)-warfarin will be designated as R-warfarin; S-(−)-warfarin will be designated as S-warfarin . The ester and the fluoro-derivatives have similar in vivo anticoagulant activity in the rat to that of S-(−)-warfarin 1 . Thus, it is possible to synthesize novel warfarin analogs that differ from racemic warfarin or its enantiomers in certain selected properties. © 1997 Elsevier Science Ltd

Genetic association between sensitivity to warfarin and expression of CYP2C9*3.

Cytochrome P4502C9 (CYP2C9) is largely responsible for terminating the anticoagulant effect of racemic warfarin via hydroxylation of the pharmacologically more potent S-enantiomer to inactive metabolites. Mutations in the CYP2C9 gene result in the expression of three allelic variants, CYP2C9*1, CYP2C9*2 and CYP2C9*3. Both CYP2C9*2 and CYP2C9*3 exhibit altered catalytic properties in vitro relative to the wild-type enzyme. In the present study, a patient was genotyped who had proven unusually sensitive to warfarin therapy and could tolerate no more than 0.5 mg of the racemic drug/day. PCR-amplification of exons 3 and 7 of the CYP2C9 gene, followed by restriction digest or sequence analysis, showed that this individual was homozygous for CYP2C9*3. In addition, patient plasma warfarin enantiomer ratios and urinary 7-hydroxywarfarin enantiomer ratios were determined by chiral-phase high performance liquid chromotography in order to investigate whether either parameter might be of diagnostic value in place of a genotypic test. Control patients receiving 4-8 mg warfarin/day exhibited plasma S:R ratios of 0.50 +/- 0.25:1, whereas the patient on very low-dose warfarin exhibited an S:R ratio of 3.9:1. In contrast, the urinary 7-hydroxywarfarin S:R ratio of 4:1 showed the same stereoselectivity as that reported for control patients. Therefore, expression of CYP2C9*3 is associated with diminished clearance of S-warfarin and a dangerously exacerbated therapeutic response to normal doses of the racemic drug. Analysis of the plasma S:R warfarin ratio may serve as a useful alternative test to genotyping for this genetic defect.

Enantiomeric separation of acidic drugs by capillary electrophoresis using a combination of charged and uncharged β‐cyclodextrins as chiral selectors

AbstractHigh resolution could be achieved for the enantiomers of acidic drugs, namely, sulindac, fenoprofen, ketoprofen, warfarin, and hexobarbital, in a buffer of pH 3 by the simultaneous addition of uncharged and charged β‐cyclodextrin derivatives. The interaction of the analytes with the anionic sulfobutyl ether β‐cyclodextrin provides the analytes with an adequate electrophoretic mobility whereas the interaction with various neutral β‐cyclodextrins generates high enantioselectivity. Five neutral cyclodextrins, the native β‐cyclodextrin, as well as methyl‐, dimethyl‐, trimethyl‐ and hydroxypropyl‐β‐cyclodextrin, were tested to enhance the enantioselectivity of the electrophoretic system. High resolution values and the shortest analysis times for the five drugs tested were achieved in a buffer made of 100 mM phosphoric acid adjusted to pH 3 with triethanolamine and containing dimethyl‐ or trimethyl‐β‐cyclodextrin in addition to sulfobutyl ether β‐cyclodextrin.

Absence of an Effect of Levofloxacin on Warfarin Pharmacokinetics and Anticoagulation in Male Volunteers

Some fluoroquinolone drug-drug interactions involving inhibition of the hepatic metabolism of agents such as theophylline and caffeine have been identified. This study was designed to investigate the potential interaction of the fluoroquinolone levofloxacin in a standard multiple-dose regimen with the oral anticoagulant warfarin. Sixteen healthy male volunteers were given a single oral dose of 30 mg warfarin sodium during a multiple-dose regimen of placebo or levofloxacin 500 mg twice daily, in a placebo-controlled, randomized, double-blind, two-way crossover design. Plasma R(+) and S(-) warfarin concentrations and prothrombin times were measured for 6 days after administration of each warfarin dose. The pharmacokinetic parameters of both enantiomers of warfarin were comparable in the absence and presence of levofloxacin, with no significant differences noted in warfarin peak plasma concentration, time to peak plasma concentration, apparent total body clearance, and terminal disposition half-life. Levofloxacin also had no significant effect on warfarin pharmacodynamics, as assessed by baseline-corrected maximum prothrombin time, time to maximum prothrombin time, and area under the prothrombin time-versus-time curve. The lack of pharmacokinetic or pharmacodynamic interaction observed in this study suggests that a clinically important effect of levofloxacin on warfarin is unlikely to occur during concurrent therapy.

Allelic Variants of Human Cytochrome P450 2C9: Baculovirus-Mediated Expression, Purification, Structural Characterization, Substrate Stereoselectivity, and Prochiral Selectivity of the Wild-Type and I359L Mutant Forms

The purpose of the present studies was to define the role of the I359L allelic variant of CYP2C9 in the metabolism of the low therapeutic index anticoagulant warfarin, by performingin vitrokinetic studies with the two enantiomers of the drug. To obtain sufficient quantities of these variants to perform kinetic studies at physiologically relevant substrate concentrations, methodology was established for the high-level expression, purification, and structural characterization of wild-type CYP2C9 and CYP2C9V1 using the baculovirus system. Both forms were expressed at levels up to 250 nmol/liter and purified in 50–55% yield to specific contents of 13–14 nmol holoenzyme/mg protein. The purified preparations were characterized by Edman degradation and electrospray–mass spectrometry. Both forms of the enzyme metabolized the pharmacologically more potent (S)-enantiomer of warfarin with the same regioselectivity; however, CYP2C9V1 exhibited a fivefold lowerVmaxand a fivefold higherKmcompared to the wild-type enzyme for this substrate. Neither form of the enzyme formed significant quantities of the (R)-warfarin phenols. Additional studies performed with prochiral arylalkyl sulfides provided confirmation of the low turnover rates catalyzed by CYP2C9V1 and demonstrated further that sulfoxide product stereochemistry did not differ significantly between the two variants. Therefore, decreased catalytic efficiency rather than a gross alteration in substrate orientation appears to be the consequence of this putative active-site mutation. The greatly decreased catalytic efficiency of the I359L variant suggests that leucine homozygotes would eliminate (S)-warfarin, and probably many other CYP2C9 substrates, at much slower ratesin vivothan individuals expressing the wild-type enzyme.

A Simplified High-Performance Liquid Chromatographic Method for Direct Determination of Warfarin Enantiomers and Their Protein Binding in Stroke Patients

A simplified method for direct determination of warfarin enantiomers by high-pressure liquid chromatography with fluorescence detection has been developed. This method involves solid phase extraction of warfarin in plasma, precolumn derivatization to form diastereoisomeric esters, and post-column reaction to discriminate each enantiomer separately. Ultrafiltration was employed in the separation of unbound warfarin enantiomers. Twelve plasma samples from six stroke patients taking warfarin regularly were analyzed. The average concentration of total warfarin was 0.47 +/- 0.17 mg/L for the S-isomer and 0.69 +/- 0.18 mg/L for the R-isomer. The average protein binding was 99.67 +/- 0.33% for S-warfarin and 99.44 +/- 0.33% for R-warfarin. This methodology provides a quick and reliable technique for determining enantiomeric protein binding of warfarin in clinical settings.

Stereochemical aspects of warfarin drug interactions: use of a combined pharmacokinetic-pharmacodynamic model.

To apply a combined pharmacokinetic-pharmacodynamic model to data from warfarin drug interaction studies. The pharmacokinetic model for warfarin enantiomers combined a common first-order absorption process with individual clearance and volume of distribution values and is based on unbound drug. The complete pharmacodynamic model comprised two components: that involving inhibition of prothrombin complex activity (PCA) synthesis described by a sigmoid maximum effect (Emax) model and that relating temporal changes in PCA to synthesis and degradation. The combined model was applied to prothrombin time and plasma concentration-time data obtained after oral administration of single doses of racemic warfarin to healthy subjects either alone or during multiple dosing with the metabolic enzyme inhibitor phenylbutazone or the inducer secobarbital. The five parameters associated with the complete pharmacodynamic model were kd (0.054 +/- 0.014 hr-1), the degradation rate constant of PCA; Cu50,S (0.0026 +/- 0.0015 mg.L-1) and Cu50,R (3.45 +/- 4.20 mg.L-1), the unbound concentrations of (S)- and (R)-warfarin required to produce a 50% reduction in PCA synthesis if administered individually; gamma (0.90 +/- 0.23), the slope parameter in the sigmoid Emax model; and td (8.2 +/- 0.3 hours), the observed delay in the onset of warfarin anticoagulant response. These findings qualitatively confirm the known potency difference between warfarin enantiomers. Furthermore, although phenylbutazone and secobarbital altered the pharmacokinetics of warfarin, these compounds do not appear to influence its pharmacodynamics. Simulation studies indicate that, after racemate administration, the continual presence of the more potent (S)-enantiomer precludes accurate assessment of Cu50,R. Analysis indicates that use of racemic (rather than enantiomer) warfarin concentration data in drug interaction studies may lead to misinterpretation of pharmacodynamic data.

Disposition of warfarin enantiomers and metabolites in patients during multiple dosing with rac‐warfarin [see comments

1. The disposition of warfarin enantiomers and metabolites has been studied in 36 patients receiving chronic rac-warfarin therapy, titrated to approximately the same anticoagulant response. 2. A stereoselective h.p.l.c. assay was employed to determine the concentrations of (R)- and (S)-warfarin, (R,S)-warfarin alcohol and (S)-7-hydroxywarfarin in plasma and 24 h urine samples. The concentrations of (R)-7-hydroxywarfarin, (S,S)-warfarin alcohol and (R)-6- and (S)-6-hydroxywarfarin were also determined in urine samples. The fractions unbound of warfarin enantiomers were determined using equilibrium dialysis. 3. Wide variability was observed in daily dose requirements (mean 6.1 mg; range: 2.5-12 mg), in plasma concentrations of (S)-warfarin (0.48 mg l(-1); 0.11-1.02 mg l(-1)), (R)-warfarin (0.87 mg l(-1); 0.29-1.82 mg l(-1)), (R,S)-warfarin alcohol (0.31 mg l(-1); 0.02-0.72 mg l(-1)) and (S)-7-hydroxywarfarin (0.25 mg l(-1); 0.07-0.37 mg l(-1)) and the percentage unbound of (S)-warfarin (0.53%; 0.29%-0.82%) and (R)-warfarin (0.54%; 0.26%-0.96%). 4. The mean plasma clearances of warfarin enantiomers were 7.5 1 day-1 per 70 kg (2.5-22.1) for (S)-warfarin and 3.6 1 day-1 per 70 kg (1.6-8.8) for (R)-warfarin. There was a significant correlation between the estimated formation clearance of (S)-7-hydroxywarfarin and the clearance of (S)-warfarin, which accounted for much of the variability in the latter.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal handling of warfarin metabolites in man

The present study examines the mechanism of renal clearance of two major warfarin metabolites, S-7-hydroxywarfarin and R, S-warfarin alcohol, in three healthy subjects who each received 1.5 mg/kg rac-warfarin alone (control) or in combination with phenylbutazone (PBZ). The unbound fraction of warfarin metabolites (fu m) was predicted using the unbound fraction of warfarin enantiomers determined, using equilibrium dialysis at 37°C and an enantioselective HPLC assay. In the control phase the unbound renal clearance (CLu R) ranged from 182 to 583 ml/min (mean, 318) for S-7-hydroxywarfarin and from 23 to 191 ml/min (mean, 96) for R, S-warfarin alcohol. For S-7- hydroxywarfarin, CLu R was much greater than the glomerular filtration rate (GFR) indicating that this metabolite undergoes net secretion in th CLu R for R, S-warfarin alcohol was less than GFR indicating that this metabolite undergoes net tubular reabsorption. The CLu R in each subject for both metabolites was notably lower during the coadministration of PBZ (39 to 62 ml/min for S-7-hydroxywarfarin and 5 to 17 ml/min for R, S-warfarin alcohol) suggesting that this drug inhibits renal secretion of these metabolites. The inhibition of renal secretion of metabolites by PBZ allows an estimation of the minimum fraction of filtered metabolite that undergoes reabsorption, being 86% to 90% for R, S-warfarin alcohol and 51% to 69% for S-7-hydroxywarfarin. The difference is probably explained by the greater polarity of S-7-hydroxywarfarin. Analysis also indicates that under control conditions secretion is a major component of renal excretion for both metabolites, accounting for 80% and 84% of metabolite entering the renal tubules for S-7-hydroxywarfarin and R, S-warfarin alcohol, respectively.

Analysis of warfarin enantiomers: Chromatographic separation of six stereoisomeric esters prepared from (−)‐(1S,2R,4R)‐endo‐1,4,5,6,7,7‐hexachlorobicyclo[2.2.1]hept‐5‐ene‐2‐ carboxylic acid

AbstractReaction of rac‐warfarin, (−)‐(1S,2R,4R)‐endo‐1,4,5,6,7,7‐hexachlorobicyclo[2.2.1]hept‐5‐ene‐2‐ carboxylic acid [(−)‐HCA] and carbodiimide reagents gave two noncyclic ketonic diastereoisomeric derivatives whereas rac‐warfarin and (−)‐HCA acid chloride with 4‐(dimethylamino)pyridine gave four cyclic hemiketal diastereoisomeric ester derivatives. The structure and stereochemistry of diastereoisomeric esters prepared from warfarin and p‐chlorowarfarin were determined from 1H‐ and 13C‐NMR spectra, mass spectra, and hydrolysis to warfarin and p‐chlorowarfarin enantiomers. The structure and stereochemistry of one of the cyclic hemiketal diastereoisomeric derivatives of warfarin are supported by an X‐ray crystallographic determination. Mechanisms for the formation of all products are proposed. © 1994 Wiley‐Liss, Inc.

Human liver microsomal metabolism of the enantiomers of warfarin and acenocoumarol: P450 isozyme diversity determines the differences in their pharmacokinetics

1. To explain the large differences in (the stereoselectivity of) the clearances of the enantiomers of warfarin and acenocoumarol (4'-nitrowarfarin) their human liver microsomal metabolism has been studied and enzyme kinetic parameters determined. The effects of cimetidine, propafenone, sulphaphenazole, and omeprazole on their metabolism has been investigated. 2. The 4-hydroxycoumarins follow similar metabolic routes and are mainly hydroxylated at the 6- and 7-position (accounting for 63 to 99% of the metabolic clearances). 3. Due to the lower Km values of R- and S-acenocoumarol and higher Vmax values of S-acenocoumarol, the overall metabolic clearances of R/S acenocoumarol exceed those of R/S warfarin 6 and 66 times respectively. 4. The metabolism of both compounds is stereoselective for the S-enantiomers, which is 10 times more pronounced in the case of acenocoumarol. 5. Except for the 7-hydroxylation of the R-enantiomers (r = 0.90; P < 0.025), the 6- and 7-hydroxylation rates of R/S warfarin do not correlate with those of R/S acenocoumarol. 6. Sulphaphenazole competitively inhibits the 7- and in some samples partly (up to 50%) the 6-hydroxylation of S-warfarin as well as the 7-hydroxylation of R- and S-acenocoumarol and the 6-hydroxylation of S-acenocoumarol (Kis ranging from 0.5-1.3 microM). 7. Omeprazole partly (40-80%) inhibits the 6- and 7-hydroxylation of R-warfarin (Ki = 99 and 117 microM) and of R- (Ki = 219 and 7.2 microM) and S-acenocoumarol (Ki = 6.1 and 7.7 microM) but not S-warfarin in a competitive manner. 8. Differences in the partial (up to 40%) inhibition of the metabolism of the enantiomers of the 4-hydroxycoumarins were also observed for the relatively weak inhibitors, propafenone and cimetidine.9. The results suggest that the coumarin ring hydroxylations of both compounds are catalysed by different combinations of P450 isozymes. The 7-hydroxylation of R/S acenocoumarol and the 6-hydroxylation of S-acenocoumarol are at least partly conducted by (a) P450 isozyme(s) of the 2C subfamily different from P450 2C9 (the main S-warfarin 7- and 6-hydroxylase).

Development and validation of a high-performance liquid chromatographic method for the quantitation of warfarin enantiomers in human plasma

An HPLC method for the quantitation of warfarin enantiomers in human plasma has been developed and validated. Baseline separation of S- and R-warfarin was achieved on a silica-bonded β-cyclodextrin column with a mobile phase of acetonitrile—acetic acid—triethylamine (1000:3:2.5, v/v/v). The detection was performed at 320 nm. The established linearity range was 12.5–2500 ng ml −1 ( r > 0.99). Tle limit of quantitation was 12.5 ng ml −1 for each enantiomer. Inter-day precision and accuracy of 12.5 ng ml −1 standards were 12.1% relative standard deviation (RSD) and +0.67% bias for S-warfarin and 9.7% RSD and +10.8% bias for R-warfarin. The low quality control samples at 37.5 ng ml −1 showed 6.9% RSD and 0.0% bias for S-warfarin, 7.2% RSD and +0.5% bias for R-warfarin. S-Naproxen was used as internal standard. Potential metabolites of warfarin were well resolved from S- and R-warfarin, the internal standard ( S-naproxen) and each other. The run time was 25 min. The silica-bonded β-cyclodextrin column showed excellent stability; over 1000 samples were injected without significant loss of performance. The column variability test showed that the method can be applied on several batches of β-cyclodextrin columns but not all the β-cyclodextrin columns were suitable for this method.