To allow the simultaneous evaluation of the interaction between sulfinpyrazone and each enantiomer of racemic warfarin, pseudoracemic warfarin (1:1 12C-R(+) and 13C-S(-)warfarin) was given to six normal subjects both before and during oral sulfinpyrazone dosing. Serial blood and urine samples were analyzed for unchanged warfarin and its metabolic products by GC/MS. A mass balance of an oral dose of pseudoracemic warfarin, containing a tracer quantity of 14C-warfarin, was carried out in one of the subjects by monitoring 14C levels in urine and feces for 15 days. Concomitant sulfinpyrazone dosing markedly increased hypoprothrombinemia, decreased clearance of (S)-warfarin, and increased clearance of (R)-warfarin. Sulfinpyrazone also decreased the urinary excretion of warfarin-related products but increased their fecal excretion by an equivalent amount. Virtually all of the administered warfarin dose could be accounted for either as unchanged drug or known metabolites. Pharmacokinetic analysis of the data suggests the following: At least four distinct enzymes (two oxidases and two reductases) are involved in the metabolism of warfarin. Sulfinpyrazone increases the hypoprothrombinemia caused by warfarin primarily by inhibition of the cytochrome P-450-mediated oxidation of (S)-warfarin, the biologically more potent enantiomer. The increased clearance of (R)-warfarin results not from induction, but from its selective displacement from plasma protein binding sites.

A B S T R A C T To evaluate the interaction of phenylbutazone with racemic warfarin or R,S-(±)-warfarin in man, S-(-)-warfarin or levowarfarin was synthesized with 13C label in the 2-position of the coumarin nucleus and added to [12C]R(+)-warfarin or dextrowarfarin to fonri a [12C/13C]pseudoracemiiate of warfarin. In six normal human subjects, a single oral dose of this "cold labeled" pseudoraceemate, 1.5 mg/kg body weight, was administered with and without a daily dosage of phenylbutazone, 300 milg orally, beginning 3 d before the warfarin dose and continuing throughout the hypoprothrombinemia. Plasma samples were obtained daily and analyzed for warfarin content and for onestage prothrombin activity. Unchanged warfarin in the plasma was fractionate(d by normal-phase, high-pressure liquid chromatography, and the enantiomorphic ratios were determined by chemical-ionizationi mass spectrometry with pentadeuteriowarfarin as the internal standard. A highly significanit augmnentation of the hypoprothrombinemia of the pseudoracemate occurred during the phenylbutazone regimen (P < 0.001) compared with pseudoracemiiic warfarin adminiistered alone. There was a highly significant increase in the plasmna clearance of dextrowarfarin (P < 0.01) and a significant decrease in the plasma clearanice of levowarfarin (P < 0.05) during the phenylbutazone regimen compared with administration of warfarin alone. It was concluded that phenylbutazonie augmented the hypoprothrombinemnia of pseudoracemic warfarin stereoselectively by inhibiting the metabolic disposition of the imore hypoprothrombinemiiic levowarfarin, yet reduced the plasma levels of pseudoPortions of this work were presented at the 37th Anntual Meeting of the Federation of Amiierican Society of Experimental Biology, Atlantic City, N. J., 10 April 1978 anid have been publishecl in abstract form in 1978. Fed. Proc. 37: 545.

To evaluate the interaction of secobarbital with racemic warfarin or R,S(+/-)-warfarin, S(-)-warfarin was synthesized with 13C-label in the 2-position of the coumarin nucleus and added to 12C-R(+)-warfarin to form a 12C-/13C-warfarin pseudoracemate. Six normal subjects received 1.5 mg/kg of this "cold-labeled" pseudoracemate. It was given with and without a daily oral dose of secobarbital, 100 mg, beginning 7 days before the warfarin and continuing throughout the hypoprothrombinemia. Plasma samples were obtained daily and analyzed for warfarin and for one-stage prothrombin activity. Unchanged warfarin in plasma was fractionated by forward-phase high-pressure liquid chromatography, and enantiomorphic ratios were determined by chemical ionization-mass spectrometry with pentadeuterio-warfarin as the internal standard. There was a reduction of the hypoprothrombinemia of the pseuoracemate during the secobarbital regimen over that on warfarin alone (p < 0.001). There was an increase in plasma clearance of R-warfarin (p < 0.05) and an increase in plasma clearance of S-warfarin (p < 0.003) during the secobarbital regimen over that on warfarin alone. It was concluded that secobarbital diminished the hypoprothrombinemia of pseudoracemic warfarin by increasing plasma clearance of the more hypoprothrombinemic S-warfarin and by increasing plasma clearance of the less hypoprothombinemic R-warfarin.

A pseudoracemic technique utilizing a seable isotope in one enantiomer was employed for the simultaneous determination of (R) and (S)-warfarin from plasma of human subjects. The assay includes high performance liquid chromatographic clean-up prior to mass spectral analysis to eliminate ion interferences from either co-administered drugs or contamination of the source. The assay is reliable, accurate and precise to within 5% at the submicrogram level.

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