The mechanism of the interaction between amiodarone and warfarin in humans.

Amiodarone decreased the total body clearance of both (R)- and (S)-warfarin in normal subjects but did not change volumes of distribution. Warfarin excretion products were quantified and clearance and formation clearance values calculated. Amiodarone and metabolites inhibited the reduction of (R)-warfarin to (R,S)-warfarin alcohol-1 and the oxidation of both (R)- and (S)-warfarin to phenolic metabolites. Inhibition of warfarin hydroxylation by amiodarone in human liver microsomes was compared with the in vivo results. In agreement, the in vitro data indicates that amiodarone is a general inhibitor of the cytochrome P450 catalyzed oxidation of both enantiomers of warfarin, but the metabolism of (S)-warfarin is more strongly inhibited than that of (R)-warfarin. These data suggest that the enhanced anticoagulant effect observed when amiodarone and warfarin are coadministered is attributable to inhibition of P4502C9, the isozyme of P-450 primarily responsible for the conversion of (S)-warfarin to its major metabolite, (S)-7-hydroxywarfarin.

Characteristics of warfarin hydroxylation catalyzed by human liver microsomes.

The oxidative biotransformation of (R)- and (S)-warfarin was studied in human liver microsomes to determine whether an in vitro model could be established that would correspond to the in vivo profile that is generally observed. The quantitative pattern of oxidized products obtained from warfarin in vitro changed dramatically as a function of substrate concentration. Apparent Km values for the formation of 4', 6, 7, and 8-hydroxywarfarin indicated the presence of two easily distinguishable subsets of human liver cytochrome P-450; a high affinity subset (Km 3-15 microM) and a low affinity subset of isozymes (Km greater than 200 microM). The high affinity subset is primarily responsible for the metabolic profile of the biologically more potent (S)-enantiomer in vivo, whereas the low affinity subset is largely responsible for metabolism of the (R)-enantiomer. Apparent Vmax values alone did not reflect the relative in vivo formation clearances of the phenolic metabolites from either antipode, because the low affinity-high capacity component masked the metabolic profile of the (S)-enantiomer. However, the rank order of intrinsic clearance, Vmax/Km, for each metabolite was in good agreement with regio- and stereoselective metabolism in vivo. This investigation highlights the need for rigorous kinetic characterization of an in vitro model before reasonable correlation can be expected with in vivo data.

The mechanism of the warfarin-rifampin drug interaction in humans.

The mechanism of the drug interaction in humans between warfarin and rifampin was investigated by monitoring the elimination kinetics and metabolic disposition of a single oral dose of pseudoracemic warfarin by GC/MS. The decrease in hypoprothrombinemia observed with concomitant administration of therapeutic doses of rifampin was accompanied by a substantial decrease in the elimination half-lives of both warfarin enantiomers. Rifampin increased the clearance of (R)-warfarin threefold and the clearance of (S)-warfarin twofold. The excretion profiles for warfarin and its metabolites in urine and feces were similar for both control and treated subjects with the exception that 4'-hydroxywarfarin (stereoselective for the (S)-enantiomer) was observed when rifampin was administered. 4'-Hydroxywarfarin is a metabolite of the drug hitherto undetected in vivo in humans. Based on formation clearance values estimated for 6-, 7-, and 8-hydroxywarfarin, rifampin appears to increase the clearance of the parent drug by induction of the cytochrome P-450 isozyme(s) responsible for aromatic hydroxylation.

The effect of sulfinpyrazone on the disposition of pseudoracemic phenprocoumon in humans.

The effect of sulfinpyrazone on the pharmacokinetics and disposition of the enantiomers of pseudoracemic phenprocoumon was assessed by analyzing serial plasma, urine, and fecal samples for parent drug and metabolites by GC/MS. Essentially all of the administered dose could be accounted for either as parent drug, known metabolites, or their conjugates. Phenprocoumon and the 7-hydroxymetabolite represented the major materials recovered. All drug-related materials excreted into the urine were extensively conjugated. Sulfinpyrazone treatment did not affect the hypoprothrombinemia produced by phenprocoumon nor did it significantly alter the plasma elimination kinetics of the individual (R)- and (S)-enantiomers. However, an apparent increased free fraction of both enantiomers in plasma and inhibition of 7-hydroxylation of (S)-phenprocoumon were observed in the presence of sulfinpyrazone. The results of this study are contrasted with those of a previous study on the interaction between sulfinpyrazone and the structurally similar coumarin anticoagulant warfarin.

Metabolic fate of phenprocoumon in humans.

Samples of urine and feces were collected daily from a normal human volunteer who had received a dose of pseudoracemic phenprocoumon [an equimolar mixture of (R)-[12C]- and (S)-[2-13C]phenprocoumon] containing a tracer dose of 10 microCi of [14C]phenprocoumon and analyzed by TLC, HPLC, and GC-MS. After 25 days, 96% of the radiolabeled material was recovered (62.8% in urine and 33.3% in feces). By isotopic dilution and comparison to the Rf values, retention times, and mass fragmentograms of synthetic standards, the metabolites of the drug were identified as the 4'-, 6-, and 7-hydroxy analogues of phenprocoumon. Virtually all of the recovered radioactivity could be accounted for by the parent drug (approximately 40%) and the three metabolites (approximately 60%). The formation of both 4'-(8.1% of administered dose) and 7- (33.4% of administered dose) hydroxyphenprocoumon was highly stereoselective, giving S/R ratios of 2.86 and 1.69, respectively. The formation of 6- (15.5% of administered dose) hydroxyphenprocoumon showed little stereoselectivity (S/R ratio equal to 0.85). The urinary excretion pattern was also confirmed in four additional healthy male subjects who received a single oral dose of pseudoracemic phenprocoumon and whose urine was analyzed by GC-MS. All the drug-related materials (both hydroxylated metabolites and parent compound) that were excreted into the urine were extensively conjugated.

Stereoselective metabolism of conformational analogues of warfarin by beta-naphthoflavone-inducible cytochrome P-450.

Previous studies have shown that the structurally related oral anticoagulants warfarin and phenprocoumon are regioselectively hydroxylated in the 6- and 8-positions by hepatic microsomes obtained from 3-methylcholanthrene (3-MC) or beta-naphthoflavone (BNF) pretreated rats. Stereoselectivity for hydroxylation is also observed and favors (R)-warfarin but (S)-phenprocoumon. The possibility that the stereoselectivity of warfarin hydroxylation is a function of the solution conformation of the drug was tested with conformationally restricted analogues. In these experiments the analogues were incubated with microsomes obtained from BNF-pretreated rats and any stereoselectivity associated with 6- and 8-hydroxylation was determined. The R enantiomer of cyclocoumarol, the cyclic ketal analogue of warfarin, was found to be selectively hydroxylated, in contrast to the S enantiomer of warfarin 4-methyl ether, the ring-opened analogue. The latter compound is known to have a preferred solution conformation similar to that of phenprocoumon. The results suggest that at the active site of BNF-induced cytochrome P-450 (R)-warfarin is metabolized in its cyclic hemiketal tautomer, a form which spatially mimics the preferred solution conformation of (S)-phenprocoumon.

A stable isotope assay for phenprocoumon and its metabolites.

A gas chromatographic/mass spectrometric assay for quantifying phenprocoumon and its 4'-, 6-, 7- and 8-hydroxy metabolites in microsomal preparations is described. This assay which uses deuterium-labeled analogs of the phenprocoumon metabolites as internal standards has a lower limit of quantitation of 20 ng ml-1. Diazomethane is used to derivatize both metabolites and parent compound yielding along with the expected 4-methoxy derivative a minor amount of the 2-methoxychromone. Resolution of the methylated metabolites is accomplished by capillary gas chromatography.

The preferred solution conformation of warfarin at the active site of cytochrome P-450 based on the CD spectra in octanol/water model system.

An octanol/water model system and circular dichroism (CD) spectroscopy have been used to study the solution conformation of warfarin in aqueous and lipid environments. Upon partitioning of (S)-warfarin from buffer pH 7.4 into octanol, the position of the absorption band due to the alpha, beta-unsaturated carbonyl chromophore shifts from 210 nm in the aqueous phase to 220 nm in the octanol phase. The shift is coupled to an increase in the molecular ellipticity of the band, suggesting the formation of a dissymmetric chromophore. Comparison of CD spectra of conformationally fixed analogues of warfarin to that of warfarin in solution suggests that the compound shifts from the open side chain keto form in the aqueous phase at pH 7.4 to the cyclic hemiketal form after partitioning into the lipid octanol phase. On the basis of these results, the hemiketal form is proposed as the preferred solution conformation of warfarin in the lipid environment of the active site of cytochrome P-450 and the relationship between solution conformation and stereoselectivity of warfarin metabolism by beta-naphthoflavone inducible cytochrome P-450 is discussed.