Stereoselective Interaction of Phenylbutazone with

[I2C!13C]Warfarin Pseudoracemates in Man

ROBERTA. O'REILLY, WILLiAM F. TRAGER, CATHERINEH. MOTLEY, andWILLIAM HOWALD,Departmenits of Medicinie, Santa Clara Valley MedicalCenter, San Jose, the University of California, San Francisco, San Francisco,California 94143; Inistitute for Medical Research of Santa Clara County,San Jose, California 95128; and Department of Pharmaceutical Science,Uniiversity of Washington, Seattle, Washingtont 58195

A B S T R A C T To evaluate the interaction of phenyl-butazone with racemic warfarin or R,S-(±)-warfarin inman, S-(-)-warfarin or levowarfarin was synthesizedwith 13C label in the 2-position of the coumarinnucleus and added to [12C]R(+)-warfarin or dextro-warfarin 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 ofphenylbutazone, 300 milg orally, beginning 3 d beforethe warfarin dose and continuing throughout thehypoprothrombinemia. Plasma samples were obtaineddaily and analyzed for warfarin content and for one-stage prothrombin activity. Unchanged warfarin in theplasma was fractionate(d by normal-phase, high-pressureliquid chromatography, and the enantiomorphic ratioswere determined by chemical-ionizationi mass spec-trometry with pentadeuteriowarfarin as the internalstandard. A highly significanit augmnentation of thehypoprothrombinemia of the pseudoracemate oc-curred during the phenylbutazone regimen (P < 0.001)compared with pseudoracemiiic warfarin adminiisteredalone. There was a highly significant increase in theplasmna clearance of dextrowarfarin (P < 0.01) and asignificant decrease in the plasma clearanice oflevowarfarin (P < 0.05) during the phenylbutazoneregimen compared with administration of warfarinalone. It was concluded that phenylbutazonie aug-mented the hypoprothrombinemnia of pseudoracemicwarfarin stereoselectively by inhibiting the metabolicdisposition of the imore hypoprothrombinemiiic levo-warfarin, yet reduced the plasma levels of pseudo-

Portions of this work were presented at the 37th AnntualMeeting of the Federation of Amiierican Society of Experi-mental Biology, Atlantic City, N. J., 10 April 1978 anid havebeen publishecl in abstract form in 1978. Fed. Proc. 37: 545.

Received for publication 19 July 1979 anid in revisedform 7 Novtember 1979.

746

racemic warfarin by greatly augmenting the metabolicdisposition of dextrowarfarin.

INTRODUCTION

The interactioni of phenylbutazone and racemiicwarfarin in man is paradoxic in that the augmentationof the anticoagulant effect is associated with a morerapid eliminationi of total warfarin (1). This effect wassaid to result from dlisplacemenit of the warfarin inplasmna from its albumin-binding sites by the morehighly bound phenylbutazone resulting in an in-creased fraction of free warfarin. Lewis et al. (2)reported a differential effect of phenylbutazoneon the enanitiomnorphs of racemiiic warfarin, S-(-)-warfarin (levowarfarin) anid R-(+)-warfarin (dextro-warfarini) (2). They found that phenylbutazone de-creased the clearanee from plasmiia of' levowarfariin,the more potent anticoagulant agent, and markedlyincrease(l the clearance of dextrowarfarin. Anotherstudy in manl on1 the interaction of phenylbutazonewith the separated enantiomniorphs showed augmiienta-tion of the hypoprothrombinemiiia and an increase inplasma conicenltrationis of levowarfarini, n1o significantalteration of the hypoprothromnbinemia ancd a miiarkeddecrease in plasmna concentrations of' (lextrowarfarin(3). To determinle simuliltaneously the fate of' bothenantiomorphs of racemiic warf:arini, the formii usedclinically, levowarfarin was synithesizedl with a stableisotope of carbon, 13C, in the 2-positioni of thecoumarin nucleus (4). By combining the [13C]levo-warfarin with an equal amount of commiiilon ['2C]dex-trowarfarin, a ['2C/'3C]warfarin pse u(loracemaite wasprepared (5). This preparationi was a(linllilistere(l tonormal huiman subjects to stud(ly the simutiltaneouisinteractioln of phenylbutazone with each enantiomilorphof racemic warfajrin.

J. Clin. Invest. (© The American Society for Clitnical Investigation, Inc. * 0021-9738/80/0(3/0746/08 $1.00Volu me 65 March 1980 746-75.3

METHODS

Subjects. Six male subjects, 18-30 yr old, were studied.All were paid volunteers who were carefully informed of thenature of the experiments and who signed written consentsin accordance with all the conditions required by federalregulation and the local Research and Human SubjectReview Committee. All were in excellent health and had nottaken any other drug during the preceding 2 mo. Eachsubject served as his own control and the order of theexperiments with warfarin alone and with warfarin plusphenylbutazone in the crossover studies was assignedrandomly. The data were analyzed by Student's t test forpaired observations; P values were determined fromprobability tables for one-tailed tests (6).

Measurement of anticoagulant effect. The one-stageprothrombin activity was determined by the method of Quick,as described previously (7), in which the test result inseconds was converted to percentage of prothrombin activityby a saline-dilution curve of pooled normal plasma.The total hypoprothrombinemic effect for each experi-ment during the control period in which warfarin wasadministered alone was determined by measuring the totalarea under the curve (AUC)I for the one-stage prothrombintime expressed in seconds on a semilogarithmic scale bythe trapezoidal rule and was expressed in arbitrary units(7). The increase in AUC-prothrombin time for each subjectwas determined for the experimental period in whichpseudoracemic warfarin plus phenylbutazone were ad-ministered.

Synthesis and preparation of ['2C/'3C]pseudoracemlate ofwarfarin. (S)-warfarin containing 90% enriched 13C ex-clusively in the C2 position was synthesized as describedseparately (4). The pseudoracemic mixture was preparedl byrecrystallization from an acetone solution containing 1.0 geach of 13C (S)-warfarin -C2- (90%) and (R)-warfarin. Thepurity of the crystalline material obtained was establishedby analysis on high-pressure liquid chromatography (HPLC),five different thin-layer chromatographic systems, and bychemical ionization mass spectrometry. Gelatin capsuleswith a 200-mg capacity, containing either 1, 2, 5, 10, 25, or50 mg of pseudoracemic warfarin in a lactose (U. S.Pharmacopeia) matrix were prepared for administration. Thebiologic authenticity of the pseudoracemate as powder incapsules was verified by comparing its hypoprothrombinemiceffect and its blood levels to that of true racemic warfarinas tablets in these same six subjects. For racemic andpseudoracemic warfarin, the mean+SEMfor AUC-prothrom-bin time were 90±+12 and 85±+10 U, respectively, an insig-nificant difference (t, 0.68, P > 0.4). The blood levels measuredas AUC-warfarin were 616+63 and 630+34 mg/liter x h forracemic and pseudoracemic warfarin, also an insignificant dif-ference (t, 0.40, P > 0.4).

Preparation of plasma specimens for measuremenit ofwarfarin. Details of the analytical methodology have beenreported separately (5). A brief description of the methodfollows. Serial plasma samples, 2 ml, were spiked with 10jig of the internal standard, pentaphenyldeuteriowarfarin,acidified with hydrochloric acid and extracted with 1,2-dichloroethane. The organic layer was extracted with 2.5 Nsodium hydroxide and discarded. The basic aqueous layerthen was acidified with hydrochloric acid and extracted with1,2-dichloroethane. An aliquot of the organic laver wastransferred to a liquid scintillation vial and evaporated to

'Abbreviations used in this paper: AUC, area under thecurve; HPLC, high-pressure liquid chromatography; Vd,volume of distribution.

dryness. The residue was taken up into mobile phaseconsisting of cyclohexane:isopropanol:glacial acetic acid, in aratio of 96:3.95:0.05 and injected onto a HPLCcolumn. TheHPLCcolumn used for separation was packed with DuPont5-Am Zorbax-CN column material (E. I. DuPont de Nemours& Co., Inc., Wilmington, Del.). Unchanged racemic warfarinhad a retention time of -9 min and was detected with a 280-nmfixed wavelength detector. Phenylbutazone emerged shortlvafter the solvent front. The unchanged racemic warfarin peakwas collected in a scintillation vial and evaporated to dryness.A second pass through the HPLC gave unchanged racemicwarfarin free from all contamination with phenylbutazone.The racemic warfarin peak was collected and the solventevaporated to dryness.

Mass spectroimetric determiniationi of enianitiomiiorphicratios atnd absolute amoutnts of w;arfarini entatntiomiorphs.Details of the mass spectral analytical procedure have beenireported separately (5). A brief description of the methodfollows. The HPLC-separatecl pseudoracemic warfarini resi-dues were reconstituted in 100 ,ul of acetonitrile. Thesolution was applied to the probe tip of the direct insertioniprobe, evaporated, and inserted into the mass spectro-meter. Mass spectral peaks at 309 (MWH ioIn of [R]-warfarin), 310 (MIH+ ion of [S]-warfarin-C2-'3C [90%]), ancd314 (NIH+ ion of pentaphenvldeuteriowarfarin) wvere inte-grated under computer-conitrolled selected ion moniitorinig.The ratio of 309:310 provided a measure of R,S-warfarin,wvhereas the ratio of 309 + 310:314 provided a measure ofthe absolute amount of pseudoracemiiic warfarini present inthe biological sample. Standlard curves for both measure-ments were accurate to <5% over the range of concentra-tions determined.

Experimletnts. Each subject received pseu(loracemiic war-farin acid, 1.5 mg/kg bodx wt, in 200-mg capsules conitaininlg1-50 mg of pseudoracemic warfarin acid to e(qual 1.5 mg/kgof drug. Each subject swallowed all the capsules allotted tohim with a full glass of water in the morning at least 2 hafter breakfast; no food was ingested for at least 2 h there-after. Blood samples were obtained by venipuncture at 0, 1,2, 4, 8, 12, 24, 36, 48, 60, 72, 84, 96, 120, 144, 168, 192, 216,and 240 h after the administration of pseudoracemic warfarin.The blood was mixed in glass tubes in a proportion of 9:1with a combiniation of three parts 0.1 M1 sodiumil citrate andtwo parts 0.1 M citric acid, and centrifugedl at 2,500 rpm for30 min at 4°C. The plasma was removed an(d stored inseveral small glass tubes at -20°C.

After a 4-wk rest period, the same subject received 1 100-mgtablet of phenvibutazone (Butazolidin; Geigy Pharmaceu-ticals, Arclslev, N. Y.), 3 times/d: before breakfast, beforedinner, ancl at bedtime. On the 4th d of the phenyl-butazone regimen, each subject received the same dose ofpseudoracemic warfarin as before; 1.5 mg/kg body vt. Thedaily regimen of phenylbutazonie was continued for theduration of the hvpoprothrombinemiiia, 6-12 cl, anid then wasdiscontinued. Venous bloodl samples were obtained at thesame times as during the control period of pseudloracemiiicwarfarin alone and were processed in the same manner. Noside effects from either drug were observed in any subject.

Each subject also received a single dose of truly racemicwarfarin sodium (Coumadin sodium; Encdo Laboratories,Inc., Garden City, N. Y.) alone, 1.5 mg/kg orally as tablets.Daily blood samples were obtained to determine the bloodconcentrations of racemic wxarfarin and the prothrombill-tillmeresponses for each subject for dlirect comiiparison of theseparameters with those obtained with pseudoracemic iwarfarinacid.

Calculatiotns. The half-life of the termninal eliminiationiphase of the log plasma warfarin from 12 to 144 h was cal-

Phenylbutazone and Warfarin 747

ctilated by the method of least squares. The slope of theterminal phase, 8, was calculated from the quotient of thefacetor 0.693 and the half-life for each sulbject. The plasmaconcentration of warfarin at time zero, Cp°, was calculated bvextension of the least s(quares line for half-life to time zero.The apparent voluime of distribution, Vd, was caleculatedfrom the (quiotient of the dose of warfarin anid plasina coni-centration at timiie zero for each stubject. The area under thecturve for the plasma concentrations of each enantiomorphof warfarin, AUC-warfarin, wvas determined froin timne 0 to240 h bv the trapezoidal ruile. The plasmiia clearance of eachenantiomorplh of wvarfarin for each subject wvas calcuilatedfromii the miiean of the prodtuct of Vd and 8 and fromii the(imiotient of the warfarin dose anid AUC-warfarin.

RESULTS

I12C]R( +)-warfarini. After a(diniistration of thewarfharin pseuidoracemn<ate alone, all subjects had de-tectabl e concentrations of dextrowarfariin in theirplasml-a at 1 h (Fig. 1). The highest concentration ofdextrowarfarin was achievedl at 2 h in one stubject,at 4 h in three subjects, at 8 h in one sul)ject, and at12 h in one subject.2 The highest meani concentrationof dlextrowarfarini in plasina occurre(d at 4 h. When thewarfarin pseudoracemiate was administered during thephenylbuitazone regimnen, all of the suibjects had de-tectable concentrations of dextrowarfarini in plasmna at1 h and had the highest imean concentration at 8 h.Thle highest concentration of (lextrowarfarin during thephenyvlbuttazonie regimeni was achieve(d at 2 h in onesubject, at 4 h in one suibject, at 8 hi in three subjects,and at 12 h in one subject. The highest mean concen-trationi of dextrowarfarin in plasmiia duiring the phenyl-butazone regimen occurred at 8 h, compared with 4 hfor dextrowarfharin alone. The mean concentration of'dextrowarfarin dturing the phenylbutazone regi mencompared witlh dextrowarfarin alone was significantlylower at 1 h (P < 0.05) and 2 h (P < 0.03). Thus, thephenylbuutazone regimein resuilted in a slower rate ofabsorption of dextrowarfarin fiom the psenidoracemate.

After the peak concentrations were reached, theplasmiia concentrations of dextrowarfarin declined l)yapp)arent first-order kinetics in all subjects for the dlura-tion of the experi menits 1)oth with and withoutphenylbutazone (Fig. 1). The mean concentrations ofdextrowarfarin with and withotut phenylbutiazonefromii 4 to 12 h were not significantly different. Theywere significantly lower (P < 0.01) from 24 to 168 hand at 216 h during the phenylbutazone regimen andat 192 and 240 h (P < 0.05). The AUC-warfalrin fordlextrowarfarin dturing the phenylbutazone regimenwas reduced in all subjects. The mean AUC-warfarin for dextrowarf'arin during the phenylbuitazoneregimen, compared with that for dextrowarfa-rin alone,

2 Extensive tables of tlhe individual data are available frointhe atithors 111on0 re(lulest.

8

4

E

CP

z

3:

LI

2

04

02

0.1

40

0u)30z0 20e 14

120

I I

48 96 144 192

1218

35

661100

2zHOURS

FIGURE 1 Blood levels of warfarin anid one-stage prothromn-bin tiines after sinigle oral doses of [12C/'3C]pseudoracemicwarfarini in six normlal subjects. Phenylbutazone, 300 mIurgdaily by mouith, was admiinistered for 3 d before the psetudo-racemic warfarin close an(d conitinutied for the dturation ofthe hypoprothromnbinemia. The lines for lhalf-life were com-

puted by the imiethod of least squtares. On the lower graphthe mean±SEMoftthe onie-stage prothrombl)in test is expressed

oni the left ordiinate in second(is logarithlmlically and on

the right or(diniate in percent of normal activity. Phenyl-btitazone markedly shortenied the half-life of R(+)warfarin,prolonge(d the half-life of S(-)warfarin, and markedlyaugmnentecl the hypoprothrombinemnia of the p)seutdoraceinate.

was reduiced to a highly significant degree (P < 0.002)(Table I).

[I3C]S(-)-warfarin. When the warfarin pseuido-racemiate was adcministered alone, all sutbjects haddetectable concentrations of levowarfarini at 1 h(Fig. 1). The highest concentration of levowarfarinwas achieved at 2 h after the adml-iinistration ofwarfarini in one subject, at 4 h ini three subjects,at 8 h in one subject, and at 12 h in one subject.The highest mean concentration of levowarfarin inplasma occurred at 4 h. When the warfarin pseudo-racemate was administered during the phenylbutazoneregimen, all of the subjects had detectable concen-

748 R. A. O'Rei1llt, W. F. Trager, C. H. Motlei , a1i(1 W. IIlvoald

.Af 1-N:

TABLE IPharmacokinetir A(alil.sis of PlasImia Con(entration (f [12CJIR(+)Warfarin aynd [13C]jS(-)V)arfarin after-

Sitngle Dose of Wr'ata rin withm and wvithotit Ph1enmIl1bmtwazo0n e*

I)rug reginmen Cp tl 2 3 ALI aC Q

Mg1liter liter /I h mg/liter X h Il/nlmin

['2C]R( + )xvarfarinlWarfarin alone 6.38+(0.39 9.0±+0.5 41.2+1.7 0.017±0.001 354+20 2.61+0.14Plus phenylbutazone 6.593+0.431 8.8±0.7 26.4+1.6§ 0.027±0.002" 210±1l9Y 4.25±0.32"

[13C]S( -)warf'arinW\arfarini alone 7.44±+0.60 7.8+0.6 30.5+.5.7 0.026±0.004 277+33 3.52±0.46Plis phenylbutazone 6.13±0.24¶ 9.3±+0.411 40.0 ±3.8C 0.01 8±0.002¶ 327±25C¶ 2.86±0.24¶

[12(,/I.-3(8 aB9-\,l f'aritl[2/ ICR,S(±+)\amfirXVarfarini alone 13.05±.0.75 8.8±0.5 36.2+±3.2 0.020_(0.002 630±)34 2.96±t0.26Pluis phenuybibtazone 12.29±0.43 9.2±0.3 33.9±1.8 0.017±0.001 528±41' 3.4 3±0.27¶

Abbreviations uise(d in this table: Cpo, plasma concentration of warfarin at timne zero, calculated by extrapolationof ,B; Vd, apparent volume of distribution, calcuilated from dose of warfarini divided by Cp°; t1/2, half-life ofxvarfarin concentrationis calculated by least squiares mnethod; ,B slope of the terminal phase of plasmaconcenltrations of xwarfarin; AUCW,, total area tinder cturve for plasma conicenitrationis of warfarin; Q, warfarinclearanice calculated fromii the mean of the product of Nd andl ,B ancl of the quotient of the warfarin dose(livided by AUC,.* XWarfarimm pseuidoracemnate, 1.5 mng/kg orally, comnlposed of 0.75 mg/kg each of ['2C]R(+)varfarin anid of['3C]S(-)warfarin; phenylbntazone, 100 m-g orally thlree times dlailv beginning 3 (d before the warfarini dosean(l conitinuinlg 10 (d after it. Mean body weight Of the six sulbjects was 75.5 kg.It test for pairedl observations.§ P < 0.001.'p < 0.01.

¶ P < 0.05.

trations of levowarfarin in plasma at 1 h and had thehighest mean concentrations at 8 h. The mean con-centration of levowarfarin durinig the phenylbutazoneregimen coinpared with that (durinig warfarin adiniiis-tration alone were significantly lower both at 1 h(P < 0.05) and at 2 h (P < 0.03). The highest concentra-tiont of levowarfarin durinig the phenylbutazone regi menwas achieved at 2 h in one sublject, at 4 h in two sub-jects, at 8 h in tw7o subjects, an-d at 12 h in one subject.The highest mean concentration of levowarfarin inplasma during the pheny-lbutazone regimen occurredat 8 h conmpared with 4 h for levowarfarin alone. Thus,thie plhenylbu-tazonie regimen apparently resuilted in aslower rate of absorption of lexowarfarin from thiepseudoracemnate.

After the p)eak concentrationis were reached, theplasimia coincenitrations of levow arfarin declinied byapparenit first-order kinetics in all subjects for thedurationi of the experimnents with and withouit phenyl-butazone (Fig. 1). The nmeati concenitrationis of levo-warfarini durinig the phlenylbutazone regimnen weresignificantly different from n%varfarin alone only at 168 h(P < 0.05). The AUCw-arfarin for levowarfarin duringthe p)heniylbutazone regimeni was increased in all sixsAbjects, auidi the differeince betwzzeen the means withand(I withouit phenyl1butazone was significant (P < 0.05)(Talbl e I).

[I2CI13iCR,S( ±)twarfarin. After adminiistration of

the warfirin pseeudoracemnate alone, the highest con-cenitration of racemiiic warfarin in plasma was achievedat 2 h in one subject, at 4 h in fOur subjects, an(d at 8 hin one subject. The higlhest miiean conicenitrationi ofracemiiic warfarin alone occuirred at 4 h. When thewarfarin psemidoracemiiate was adminiistere(d dcuringthe phenll butazone regimen, the higlhiest concentra-tion of racemic warfarin in plasmna was achieved at2 h in one sulbject, at 4 h in two subjects, at 8 h intwo subhjects, and at 12 h ini one subject. The highestmeani concentration of racemic warfarin dutring thephenylbuitazone regimen occiurred at 8 h, comparedwith 4 h for warfarin alone. The meani concentrationof racemic wvarfarin (Iliritng the phenylbutazoneregi men compared with racemic xwarfarin alonie wassignificantly lower both at I and 2 h (P < 0.05). Thus,the phenyllbitazone regimnen resuilted in a slower rateof absorption of racemic warfarin from the pseudo-racemnate.

After the peak concenitrations wvere reached, theplasmat concentrations of racemnic warfarin declined byapparenit first-ordler kinetics in all suibjects for thedIuration- of' the experimiients 1oth with andl withoutphenylbuitazone. The imean concentrations of racemicwvarfarin durinig the plhenyll)utazone regimen weresignificantly different fromn adminiistration of warfarinalonie only at 60 and 84 h (P < 0.02). The total AUC-wvarfarin for racemic warfarin duiiring the phenN l-

Phenylbmitazotne and W1'arfaritm 749

butazone regimen was reduced for all subjects. Themean AUC-warfarin for racemic warfarin during thephenylbtutazone regimen compared with that forracemnic warfarin alone was reduced to a highlysignificant degree (P < 0.01) (Table I).

One-stage prothrombin activity. After administra-tion of the single dose of the warfarin pseudo-racemiate alone, all six subjects had detectable hypo-prothrombinemia after 12 h (Fig. 1). The most markeddepression occurred 48-96 h after the warfarin dose,and the return to 100% of normal activity occurred144-240 h after it. The mean of the lowest one-stageprothromibin activity occturred at 60 h. The magnitudeof the AUC-prothrombin time for the one-stageprothrombin activity varied fromi 56 to 121 U; thenean±SEM for AUC-prothrombin time was 85± 10 U.

After adiministration of the warfarin pseudoracemiateduring the phenylbutazone regimen, all six subjectshad detectable hypoprothrombinemia after 12 h. Themost mlarked depression occurred 72-120 h after thewarfarin dose, and the return to 100% of normalactivity occurred at 240 h in three subjects and oc-curred after 240 h in the other three subjects. Themean of' the lowest one-stage prothroimbin activityoccutrred 84 h after the warfarin dose during thephenylbutazone regimeni, coimpared with 60 h afterthe warfarin dose alone. The difference between themean of' the hypoprothromnbinemnia for warfarin aloneand during the phenylbutazone regimen in the first36 h after the warfarin dose was not significant, from48 to 60 h was significant (P < 0.02), from 72 to 216 hwas highly significant (P < 0.01), and was not sig-nificant at 240 h. The AUC-prothrombin time for theone-stage prothrombin activity increased markedly inall six subjects. The mean±SEM was 158±13 U,compared with a mean AUC-prothrombin time forwarfarin alone of 85± 10 U, a highly significant augmen-tation (P < 0.001) of the hypoprothrombineimia duringthe phenylbutazone regimeni.

Pharmnacokinetic analysis of plasmna concentrationsof dextrowarfarin. A pharimacokinetic analysis wasundertaken on the plasma concentrations of [12C]R(+)-warfarin, or dextrowarfarin, after single doses of war-farin pseudoracemate with and without the phenyl-butazone regimen (Table I). The means of plasmaconcentration at time zero and of Vd for warfarinalone were not significantly different from those forwarfarin during the phenylbutazone regimen. Themean Vd was 9.0 liters for the warfarin alone and 8.8liters for warfarin during the phenylbutazone regimen,or 11.9 and 11.7% of the average body weight,respectively. The mean±SEM for the half-life ofdextrowarfarin was 41.2±+1.7 h for warfarin alone and26.4±1.6 h for warfarin during the phenylbutazoneregimen, a highly significant difference (P <0.001).

The mean±SEMarea under the curve for the plasimaconcentration of warfarin, AUC-warfarin, was 354±220mg/liter x h for warfarin alone and 210±19 img/literx h for warfarin during the phenylbutazone regiinen,a highly significant difference (P <0.01). The imeanplasmna clearance of dextrowarfarin was 2.61±+0.15inl/nin for warfarin alone and 4.25±0.32 iml/mnin forwarfarin during the phenylbutazone regimen, a highlysignificant increase in dextrowarfarin clearance (P< 0.01).

Pharmtacokinetic analiysis of plasmna concentratio().sof levowarfarin. A pharmacokinetic analysis wasundertaken on the plasma concentrations [13C]S(-)-warfarin, or levowarfarin, after single doses of thewarfariin pseudoraceimate with and withouit thephenylbutazone reginen (Table I). The meani Vd was7.8±0.6 liters for warfarin alone and 9.3±0.4 liters forwarfarin during the phenylbutazone regiinen, a highlysignificant difference (P <0.01), anid were 10.3 and12.3% of body wt, respectively. The mean SEMfor half-life of levowarf:arin was 30.5±+5.7 h for warfarinalonie and 40.0±3.8 h for warfarin during the phenyl-butazone regimen, a significant difference (P < 0.05).The mean+SEM for AUC-warfarin was 277±33 mg/liter x h for warfarin alone and 327±25 mg/liter x hfor levowarfarin durinng the phe nylbutazone regi men,a significant differenice (P < 0.05). The mean±SEMforplasina clearance was 3.52±0.46 ml/min for warfarinalone and 2.86±0.24 ml/min for warfarin during thephenylbutazone regimnen, a significant decrease inlevowarfarin clearance (P < 0.05).

Pharinacokinetic analysis of plasma concentrationsof pseudoracemic warfarin. A pharmacokinetic analy-sis of pseudoraceinic warfarin after single doses of thewarfarin pseudoraceinate was undertaken on the sumof the plasma concentrations of [12C]R(+)warfarin andI[3C]S(-)warfarin for each subject at every time tested.The means for Vd were not significantly different forwarfarin alone and for warfarin during the phenyl-butazone regimen. The mean Vd was 8.8 liters forpseudoracemic warfarin alone and 9.2 liters for pseudo-racemnic warfarin during the phenylbutazone regimen,or 11.7% and 12.2% of body wt, respectively. Themean±SEM half-life was 36.2±3.2 h for pseudo-raceinic warfarin alone and 33.9±1.8 h for pseudo-racemic warfarin during the phenylbutazone regimen,an insignificant difference. The mean±SEMfor AUC-warfarin was 630±34 mg/liter x h for pseudoracemicwarfarin alone and 528±41 mg/liter x h for pseudo-racemic warfarin during the phenylbutazone regimen,a significant difference (P < 0.01). The mean±SEMfor plasma clearance was 2.96±0.26 ml/min for war-farin alone and 3.43±0.27 ml/min for warfarin duringthe phenylbutazone regimen, a significant increase inpseudoracemic warfarin clearance (P < 0.05).

750 R. A. O'Reilly, WV. F. Trager, C. H. Motley, and W. Hotvald

DISCUSSION

The authenticity of pseudoracemic warfarin in bothits synthesis and preparation was validated by directcomparison with commercial racemic warfarin in allthe research subjects. The total hypoprothrombinemiceffect was evaluated by comparison of the meanAUC-prothrombin time for pseudoracemic warfarinand for racemnic warfarin. The mean difference wasinsignificant. Similarly, there was no significant dif-ference in the AUC of the blood concentrationsachieved when pseudoracemic and racemic warfarinwere comipared. The hypoprothrombinemic effect ofpseudoracemic warfarin with daily phenylbutiazoneadministration was 186% of the control value, whichis similar to the augmentation by phenylbutazonepreviously reported for racemic warfarin in normnalsubjects (1, 2). Thus, the artificial admixture ofsynthetic [1'3C]levowarfarin and [12C]dextrowarfarinas pseudoracemic warfarin behaved pharmiiaeologicallylike synthetic racemiiie warfarin.

Although the interaction of warfarin and phenyl-butazone has been previously studied with racemicwarfarin (1, 2) and with the separated enantiomnorphs ofwarfarin (2), no previous data have been published onthe fate of each enanitiomorph in racemic warfarinduring the interactioni. This approach is importantbecause of the widely divergent effects of phenyl-butazone on the enantiomorphs of warfarin. Lewis andTrager (2) found in two subjects that phenylbutazonespeeded up the metabolic disposition of dextrowarfarinand slowed down the metabolic disposition oflevowarfarin in plasma. They studied the urinarymetabolic products of racemic warfarin with and with-out phenylbutazone in two other subjects andl con-eluded that the effect of phenylbutazone on thewarfarin enantiomorphs administered together wassimilar to that on the separated enantiomorphs (2). Ourstudy has confirmed that report by direct measuremenitof the warfarin enantiomiiorphs (luring their simultanieousadministratioin as a pseudoracemate. The synthesis ofone enantiomorph of a racemate with 13C to prepare a[I2C/13C]pseudoracemate may have wide application toother racemic drugs.

Phenvlbutazone seemied to slow the gastrointestinialabsorption rate of both dextrowarfarin and( levowar-farin, as well as pseudoracemic warfarin. This effect ofphenylbutazone on the absorption rate of warfarinihitherto has not been reported. These findings prob-ably were not observed before because previousinvestigations of this drug interactioni didl not inclti(leearly blood samplinig. Racemic warfarin is completelyabsorbed in normal subjects, whereas the oralanticoagulant dicumarol is not (8). The absorptioni ofracemic warfarin was not affected even by diseases

associated with severe gastrointestinal malabsorption.Cholestyramine in man interfered with the absorptionof racemic warfarin causing it to appear in the stool (9),and also interfered with the absorption and subsequenthypoprothrombinemic effect of racemic phenprocoumon(10). Barbiturates did not affect the absorption ofracemic warfarin, but did reduce the absorption ofdicumarol (11). Antacid therapy had no effect on theabsorption of racemic warfarin (12). The simultaneousingestion of food interfered with the rate but not theextent of absorption of racemic warfarin in man (13),and actually enhanced the absorption of poorlyabsorbable dicumarol in one study in man (14). Thus,the impact of phenylbutazone on the absorption ofpseudoracemic warfarin is similar to that of food:reduction in the rate, but not the extent of racemicwarfarin absorption, and no effect on its bioavailabilityor hypoprothrombinemic effect.

Racemnic warfarin acid is the rodenticidal form ofthe drug, whereas racemic warfarini sodium (Coumadin;Panwarfini, Abbott Laboratories, North Chicago, Ill.)or raceirric warfarin potassiumi (Athrombin-K, ThePurdue Frederick Company, Norwalk, Conn.) is theformn used clinically. All the ['2C]- or [13C]warfarinpreparations used in this study were warfarin acids.Perhaps, the effect of phenyll)utazone on the absorp-tion rate of pseudoracemiiic warfarin acid, dextro-warfarin acid, and levowarfarin acid wouild not haveoccurred with the formii used clinically, racemnicwarfarin soditumii. However, this possibility is un-likely both becautse of' conversion to warfarin acid oncontact with the acid content of the stomiach, andbecautse previotis sttudies in man comnparinig tablets ofracemnic warfarin acid to racemic warfairini sodiumlnshowed no significant differences in the gastrointestinalabsorptioni rate of the drug, bioavailability, timie ofpeak 1)lood levels, AUC-warfarin, AUC-prothrombintime, Vd, or half-life (8, 15).

The following imechaniismi cani l)e proposecl for theinteractioni of phenylbutazone with racemic Zvarfarin.Dextrowarf'arini undergoes inetabolic tranisformiiationpriim1arily by reduction of the ketonie fuinctioin ofthe acetonyl side chalinl to two or miiore secondaryalcohols, which are excreted mncainly by the kidneyinto the uirinie (16). Levowarfarin uindergoes inetal)olictranisformiiation primarily by oxidation via rinig hy(lrox-vlation of the coumlnarini nuicleuis to 7-hv,droxvlevowar-f'arin, which is excreted by the liver mainilv into thebile andl evenituially into the stool (17). It is proposedthat the acetivity of the enzymnes controlling the ringoxidationi of' levowarfhiri minav l)e inhilitedl l)y phleniyl-butazone. Sttudies witlh single enantionmorphs of war-farim sutpport this stuggestion (2). This inhibitionwouild imiipair the total body clearanice of levowarfarilland lead to hiigher blood levels andl a longer half-life

Pheqibtylbttazone an d Warfarin 751

in plasma for unchanged levowarfarin. It is also pro-posed that the activity of the enzymes controlling theside-chain reduction of dextrowarfarin perhaps is in-creased via enzymatic induction by phenylbutazone.This enzymatic stimulation would increase the totalbody clearance of dextrowarfarin and lead to lowerblood levels and a shorter half-life in plasma forunchanged dextrowarfarin.

The greater quantity in the body of the more potentanticoagulant levowarfarin, even with a lesser quantityof total warfarin, would hinder the synthesis of thevitamin K-dependent clotting factors more com-pletely and for a longer time in the presence ofphenylbutazone (18). This lessened synthesis of clot-ting factors would lead to a greater hypoprothrom-binemic effect with both racemic and pseudo-racemic warfarin. However, the alterations in the totalbody clearances of the enantiomorphs of warfarin byphenylbutazone, particularly that of levowarfarin, aremodest compared with the marked augmentation of thehypoprothrombinemia in every subject tested. Thesedata suggest that other factors contribute to theinteraction.

A phenylbutazone-induced increase in the freefraction of racemic warfarin was reported by Aggelerand co-workers (1) and confirmed by Lewis et al. (2).The importance of this alteration of albumin-boundwarfarin by phenylbutazone was observed by Tille-ment et al. (19). They found that phenylbutazoneincreased the free fraction of the three coumarinanticoagulants tested (acenocoumarol, ethyl bis-coumacetate, and warfarin) but had no effect on thatof the indanedione anticoagulants tested (fluoro-phenindione and phenindione). They correlated thesebinding data with the marked hypoprothrombinemicaugmentation of coumarin anticoagulants and theabsence of this interaction with indanediones (19).Our data showed a highly significant increase in theVd of levowarfarin, which is consistent with anincrease in the free fraction of levowarfarin. Thehalf-life of oral pseudoracemic warfarin administra-tion by the one-compartment analysis reported herein,36 h, is similar to administration of truly racemicwarfarin intravenously by a two-compartment openmodel, 35 h (20). It is also possible that the inter-action results from a direct effect of phenylbutazoneon the synthesis or the degradation of the vitaminK-dependent clotting factors. This possibility seemsunlikely because phenylbutazone has no hypopro-thrombinemic action by itself and has no effect onthe degradation rate of clotting factors as evidencedby comparable prolongation of the prothrombintime for the first 48 h after the warfarin dose withand without phenylbutazone (21). Thus, the most likelyexplanation for the interaction is a complex combina-

tion of effects: protein displacement and stereoselec-tive metabolic changes.

The study reported herein demonstrated a stereo-selective interaction of phenylbutazone on pseudo-racemic warfarin. Other studies using separatedenantiomorphs of racemic warfarin also demonstrateda stereoselective interaction with phenylbutazone:decreased clearance of levowarfarin and increasedclearance of dextrowarfarin (3). Phenylbutazone aug-mented the hypoprothrombinemia of levowarfarin buthad little or no effect on that of dextrowarfarin.These findings suggest that the interaction of racemicwarfarin and phenylbutazone may be lessened ifracemic warfarin were replaced by dextrowarfarin forlong-term therapy. Thus, long-term therapy with oralanticoagulants could become more stable and lessdangerous. Substantiation of this possibility requiresfurther detailed investigations to clarify the toxicityof dextrowarfarin at higher dosage and any significantinteraction with phenylbutazone at those doses.

ACKNOWLEDGMENTSThe authors are grateful to Mrs. Anne Goulart, Ms.Susan Nutter, and Mrs. Marjorie Kline for their experttechnical, editorial, and typographical assistance, respectively.

This work was supported in part by grants GMS22860-04,HL 08058-15, and Research Career Development AwardGMU-2111 (Dr. Trager) from the U. S. Public HealthService.

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