The influence of a graded dose schedule of aminoglutethimide on the disposition of the optical enantiomers of warfarin in patients with breast cancer

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1 Cancer Chemother Pharmacol (1986) 17: ancer hemotherapy and harmacology Sprnger-Verlag 1986 The nfluence of a graded dose schedule of amnoglutethmde on the dsposton of the optcal enantomers of warfarn n patents wth breast cancer P. E. L~nnng l*, P. M. Ueland 2, and S. Kvnnsland 1 1 epartment of Oncology, Haukeland Sykehus, Unversty of Bergen, N-5000 Bergen, Norway 2 Clncal Pharmacology Unt, Haukeland Sykehus, Unversty of Bergen, N-5000 Bergen, Norway Summary. The pharmacoknetcs of the optcal enantomers of warfarn (R-warfarn and S-warfarn) were nvestgated n patents treated for breast cancer wth amnoglutethmde (G). The patents receved 125 mg G b..d. (.e., low-dosage regmen); 250 mg G q..d, together wth cortsone acetate (. e. hgh-dosage regmen); or an escalatng dose schedule was followed (.e. low-dosage regmen followed by hgh-dosage regmen). The pharmacoknetcs for R-warfarn and S-warfarn were determned before ntaton of G treatment and agan after 2, 4, or 8 weeks of contnuous G treatment. The plasma clearance for both enantomers showed a moderate ncrease (mean 41.2%) n patents recevng the low G dose, whereas n patents treated accordng to the hgh-dosage regmen a marked ncrease (mean 90.8%) was observed. There was a correspondng reducton n warfarn half-lfe, and no alteraton n dstrbuton volume. These effects on the warfarn pharmacoknetcs appeared after 14 days of G treatment, and after ths tme pont there was no further ncrease n warfarn clearance. Notably, the effect of G on warfarn knetcs was the same for both enantomerc forms of warfarn. These data show that there s a dose-response relatonshp between G dose and nducton of warfarn metabolsm. Introducton mnoglutethmde (G) nhbts the growth of hormonedependent metastatc breast carcnoma, and s currently used n the pallatve treatment of advanced breast cancer n postmenopausal women [21]. The therapeutc effcency has been attrbuted to the fact that G nterferes wth estrogen bosynthess. The man target of G s probably aromatase, the enzyme system responsble for the converson of androgens to estrogens. Ths pathway s the major source of estrogens n postmenopausal women [22]. The conventonal dosage regmen for G s 250 mg q..d., combned wth hydrocortsone [8]. Ths drug sche- dule s referred to as a "hgh-dose schedule" n ths study. In vvo studes have shown that a lower dose G drug schedule (125 mg b..d.) exerts a pronounced nhbtory effect on the aromatase system [3], and low-dose G wthout hydrocortsone has been reported to produce a decrease n plasma estrogens smlar to that obtaned wth conventonal hgh-dose therapy [4, 26]. Ths regmen s referred to as a "low-dose schedule" n ths study. Low-dose treatment wth G s now undergong clncal trals as an alternatve treatment for breast cancer [5, 14, 24]. G gven as hgh-dose treatment enhances the metabolsm of several drugs [10, 12, 13, 21], suggestng that G s an effectve nducer of the mxed functon oxdase system of the lver. Inducton of drug metabolsm n lver may form the bass for nteractons between G and other drugs gven to patents wth breast cancer. We have prevously reported on the clncal mplcatons of enhancement of warfarn metabolsm by hgh-dose G treatment [11]. Whether low-dose G treatment has a smlar effect on drug metabolsm s an open queston. The antcoagulant drug, warfarn, has an asymmetrc carbon atom and exsts n two somerc forms, R- and S- warfarn. The clncal form s a racemate, but S-warfarn s more effectve as an antcoagulant than the R-form [6]. One mportant pont s that warfarn undergoes stereoselectve hydroxylaton n the lver. S-Warfarn undergoes faster elmnaton than the R-somer because of dfferences n metabolsm. Some drug nteractons nvolvng warfarn are stereoselectve and affect the S-enantomer only [9, 17]. The dfferental metabolc fate of R- and S-warfarn has recently been exploted by usng the plasma half-lfe of the R-and S-enantomers as a measure of nducton of the hepatc mxed functon oxdase system [9, 17]. The am of the present nvestgaton was to characterze the ablty of G to nduce drug metabolsm by usng R- and S-warfarn as a clncal probe. The followng aspects of G/warfarn nteracton were addressed: (1) dose dependency, (2) tme dependency and (3) possble stereoselectvty. * Ths work was supported by grants from the Norwegan Cancer Socety and the Norwegan Socety for Fghtng Cancer. The authors gratefully acknowledge the skllful techncal assstance of Mr Hallvard Bergesen and Mrs Gry Kvalhem * PEL s a Research Fellow of the Norwegan Cancer Socety Offprnt requests to: P. E. Lonnng Patents and methods Patents. Nne women recevng G as a treatment aganst metastatc breast cancer were ncluded n ths study. Ther mean age was 62 years (range years), and ther mean body weght 71.3 kg (range kg). Two patents (W. B. and B. S.) were moderate smokers (5-10 cga-

2 178 rettes per day). part from G, drugs known to affect hepatc drug metabolsm were not ngested by the patents partcpatng n the study. ll patents had normal platelet counts, thrombotests, bleedng tmes, and serum transamnase actvtes. ll patents gave nformed consent to partcpaton n the study. Ultrasonography of the lver was normal n all nne patents. G dose schedules. Patents receved ether 125 mg G b..d. (low-dosage regmen) or 250 mg G q..d, n combnaton wth 25 mg cortsone-acetate b..d. (hgh-dosage regmen). Four patents receved low-dose G for the frst 4 weeks of treatment and two patents for 8 weeks before ntaton of the hgh-dosage regmen. Two patents receved treatment wth hgh-dose G from the start of drug therapy, and these two patents receved 50 mg cortsone acetate b..d, for the frst 2 weeks, followed by 25 mg b..d. [8]. Protocol and blood samplng. The plasma half-lfe, clearance, and dstrbuton volume of R- or S-warfarn were determned under the followng condtons: a) Before ntaton of treatment wth G. Ths s referred to as phase 1. b) fter treatment for 2 or 4 weeks. Ths ntermedary perod of treatment s referred to as phase 2. The patents receved ether low- or hgh-dose treatment. c) fter treatment wth G for a total tme of 8 weeks. Ths s defned as phase 3. Some patents receved lowdose treatment for the frst 4 weeks, followed by hgh-dose treatment for the next 4 weeks, whereas others were treated wth the same dose (hgh or low) throughout the perod. In one patent (. F.) G therapy had to be dscontnued after less than 8 weeks, because of rapd progresson of the dsease state. Each patent was gven the same warfarn enantomer throughout the study. The drugs were dssolved n water, and gven as a sngle oral dose of ether 0.20 mg S-warfarn/kg body weght or 0.33 mg R-warfarn/kg body weght. The dose of S-warfarn was smaller than that of the less potent antcoagulant, R-warfarn, to avod an untoward antcoagulant effect. The dose was always admnstered at 8 a. m. after an overnght fast. Food or other drugs (G or hydrocortsone) were allowed 2 h after warfarn ngeston. Blood samples for warfarn determnaton were taken at 0, 2, 4, 6, 9, 12, 24, 36, 48, 72, 96 and 120 h after ntake of the R-enantomer. The same protocol was used for the S-enantomer, except that the last blood sample was collected after 96 h. In some patents addtonal blood samples were collected, for example when venepunture was done for other reasons or f routne sample(s) were accdentally lost. Blood samples for G determnatons were collected 0, 6, 9, and 12 h after admnstraton of warfarn, and the mean G concentraton value calculated. ll blood samples were allowed to coagulate for 30 mn to 1 h. Then serum was prepared by centrfugaton, and serum samples stored n darkness at -20 C untl analyss. Chemcals and drugs. cetontrl (HPLC-grade) was purchased from Rathburn Chemcals, Ltd, Peeblesshre, Scotland. OS hypersl 34tm mcropartcle medum for reversed-phase chromatography was from Shandon Southern Products Ltd, Cheshre, UK., and Pellguard LC 18 (40 lxm) from Supelco, Inc., Bellefonte, Pa US The analytcal columns were slurry-packed wth hypersl at 9000 ps, usng a Shandon column packer. The optcal somers of warfarn were kndly suppled by Chemoswed Co., Malmo, Sweden. The purty of the enantomers was evaluated by optcal rotaton ([alfa] of for the R-somer and for the S-somer). G was a gft from Cba-Gegy, Basel, Swtzerland. Both R- and S-warfarn and G were pure accordng to HPLC analyss (see below). rug analyses. Warfarn (R- and S-enantomers) was analyzed by an HPLC assay descrbed prevously [25]. Brefly, samples of deprotenzed serum were subjected to chromatography on a reversed-phase (Hypersl) column eluted wth 23% acetontrl n 100mM ammonum formate, ph 3.5. The flow rate was 2 ml/mn. The absorbance was routnely recorded at 305 nm usng a photodode array detector. The retenton tme was 2.6 mn for both somers. The dentty of the warfarn peaks was confrmed by recordng the spectra at the up and down slope and at the top of the peak. ll samples were analyzed n duplcate. G and ts major plasma metabolte, N-acetyl-G, were analyzed as descrbed recently [23]. Serum was deprotenzed by a mxture of acetontrl and perchlorc acd. The chromatographc system was the same as that used for warfarn analyss, except that the acetontrl concentraton of the moble phase was reduced to 11% and the absorbance was routnely recorded at 242 nm. Pharmacoknetc calculatons. These were performed as recommended by The mercan College of Clncal Pharmacology [1]. Clearance (CL) for warfarn was calculated accordng to the formula f CL- UC where f = fracton of the dose absorbed, = dose of drug, and UC the area under the serum concentraton curve from tme zero to nfnty. The value for f s regarded as 1 snce warfarn s almost completely absorbed after oral admnstraton and there s no detectable frst-pass metabolsm [16, 181. UC was determned by the trapezodal rule from tme zero to the tme correspondng to the last sgnfcant value for serum warfarn. The resdual area was added and calculated by extrapolaton to nfnty after loglnear least square regresson analyss of the termnal part of the curve. The apparent volume of dstrbuton (Vz) was calculated by equaton f vz ~z UC' where )~z s the termnal rate constant. Ths constant was estmated from the termnal elmnaton phase usng log lnear regresson analyss. Statstcal analyss. Trends were analyzed usng Spearman rank correlaton coeffcents (Rs). Results Warfarn elmnaton curves and curve ft The warfarn serum concentraton curves obtaned after oral admnstraton of the R- or S-enantomer to two pa-

3 179 I ,m o Phase 1 I Phase 2 = Phase l I Phase 2 r- ~1.o ~m g II n ~ Phase 3 - a I c ] Phase 3 o.i Tme (hours) 0.1 I I I Tme (hours) I Fg. 1. Serum concentraton curves for R-warfarn n a patent (M. V.) before start of G therapy (phase 1), after recevng lowdose G for 4 weeks (phase 2), and after recevng hgh-dose G for the next 4 weeks (phase 3) Fg. 2. Serum concentraton curves for S-warfarn n a patent (W. K.) before start of G therapy (phase 1), after recevng lowdose G for 4 weeks (phase 2), and after recevng hgh-dose G for the next 4 weeks (phase 3) Table 1. Pharmacoknetc parameters of R-warfarn before and durng treatment wth G a Patents Phase of uraton osage G n serum treatment of treatment regmen b (p~g/ml) (weeks) Warfarn Clearance tl/~ (h) Vz (l/h) (ml h -~ kg- ~) (1) (ml/kg).f low G.J low low J hgh hgh M.V low hgh W.B low hgh a R-Warfarn was gven as a sngle oral dose before ntaton of treatment (phase 8 weeks of G treatment (phase 3) b The patents were trated accordng to the low- or hgh-dosage regmen 1), after treatment for 2 or 4 weeks (phase 2), and after tents n phases 1, 2 and 3 of G treatment (see expermental secton), are shown n Fg. 1 and 2. The curves were sometmes bphasc (6 out of 26), wth an ntal short phase followed by an elmnaton phase, consstent wth a two-compartment model. The alfa-phase was often masked by a delayed absorpton, gvng a monophasc serum concentraton curve, and a one-compartment model gave the best curve ft. To treat all data equally, model-ndependent calculatons were chosen throughout ths study. Warfarn knetcs and G dose Clearance, half-lfe and dstrbuton volume were determned for ether R- or S-warfarn n nne patents treated wth G (Tables 1 and 2). Low-dose G treatment of patents nduced an ncrease n warfarn clearance after 4 weeks of treatment (phase 2 versus phase 1), whch vared markedly from one patent to another (range 15%-103%). There was a correspondng decrease n warfarn half-lfe n serum, whereas dstrbuton volume was not affected. Notably, the same response was observed for R- and for S-warfarn. Four patents were treated wth low-dose G for up to 8 weeks (phase 3). In these patents, there was no further ncrease n clearance of R- or S-warfarn (Tables 1 and 2). Hgh-dose G treatment was gven to two patents from the ntaton of therapy. fter 2 weeks there was a marked ncrease (57% and 65%) n clearance of both R- and S-warfarn, wth a correspondng reducton n halflfe. No further effect was observed when the G therapy was contnued for 8 weeks. Four patents treated wth lowdose G for 4 weeks showed a mean ncrease n warfarn clearance of about 39%. The hgh-dose treatment n these patents nduced a further ncrease n clearance, to a mean

4 180 Table 2. Pharmacoknetc parameters of S-warfarn before and durng treatment wth G a Patents Phase of uraton osage G n serum Warfarn treatment of treatment regmen b (~g/ml) (weeks) Clearance tl/2 (h) Vz (I/h) (ml h -~ kg -~) (1) (ml/kg) E.E low low K.O hgh hgh W.K low hgh B.S low hgh a S-Warfarn was gven as a sngle oral dose before ntaton of treatment (phase 8 weeks of G treatment (phase 3) b The patents were treated accordng to the low- or hgh-dose regmen 1), after treatment for 2 or 4 weeks (phase 2), and after value 107% above that obtaned n phase 1. Smlar results were obtaned for R- and S-warfarn somers (Tables 1 and 2). G n serum Mean G serum concentratons of patents recevng lowdose ( ~tmg/ml) or hgh-dose G drug schedules ( ~tg/ml) were as reported n prevous studes [12l. The serum concentratons n patents treated accordng to the low-dosage regmen for 4 weeks showed no correlaton (Rs = 0) wth the ncrease n warfarn clearance. Nor dd the data obtaned for patents treated wth hghdose G at tme 8 weeks after ntaton of therapy reveal any correlaton between G concentraton and ncrease n warfarn clearance (Rs = -0.32). In one patent (G. J.) who receved low-dose G and showed a remarkably hgh ncrease n warfarn clearance, the amount of G n serum equalled that measured n serum from patents recevng hgh-dose G (Tables 1 and 2). scusson In the present paper we nvestgated the ablty of G to nduce hepatc drug metabolsm, usng the warfarn enantomers as a clncal probe. The serum clearance of warfarn reflects the actvty of the warfarn-metabolzng enzyme system, and can therefore serve as an ndex of the effect of G treatment on warfarn metabolsm. The pharmacoknetc parameters of R- and S-warfarn were determned after oral admnstraton. Ths was consdered adequate, snce both warfarn enantomers are well absorbed [9, 18] and do not undergo sgnfcant frst-pass metabolsm [16]. The pharmacoknetcs of warfarn admnstered IV have been reported to be best ftted by a two-compartment open model [19]. We observed a monoexponental serum curve n most patents (Fgs. 1 and 2). Ths could be explaned by the fact that the dstrbuton phase, whch subsdes wthn 6 h [19], s obscured by the peak concentraton occurrng 3-6 h after PO admnstraton [18]. The values we obtaned for clearance, termnal half- lfe, and dstrbuton volume for R- and S-warfarn n phase 1 (Tables l and 2) are n accordance wth those publshed by others [7]. We have prevously demonstrated that treatment wth conventonal hgh-dose G for 4-8 weeks nduced a marked ncrease n the clearance of warfarn racemate [11]. In the present paper t s demonstrated that the ncrease n warfarn metabolsm nduced by G s fully developed wthn 14 days (Tables 1 and 2). Other studes on nducton of drug metabolsm n man have shown a smlar tme dependency [20]. Furthermore, the ntroducton of the lowdose G treatment as a therapeutc regmen n the management of breast cancer [24] warranted the nvestgaton of the dose requrement of the nductve effect. We observed that low-dose G nduced a sgnfcant ncrease n warfarn clearance, whch was further ncreased by hghdose treatment (Tables 1 and 2). Ths shows that wthn the therapeutc range there s a dose-response relatonshp for the nductve effect of G. Several other nducers of the hepatc mxed functon oxdase system, lke barbturates and rfampcn, have been shown to produce a dose-dependent nducton [2, 15]. We observed no sgnfcant relaton between the G serum concentraton and ncrease n warfarn clearance. Ths may be explaned by the large nterndvdual varatons n serum G level and the lmted number of patents studed. G nduced the metabolsm of the two warfarn enantomers to the same extent (Tables 1 and 2), showng that no stereoselectvty exsts. Our data are n accordance wth an effect of G on a common metabolc pathway, e.g. 6-hydroxylaton, whch s shared by both enantomers [9]. However, an addtonal effect on 7-hydroxylase cannot be excluded. In concluson, the possble mplcatons of the fndngs reported are as follows: 1) moderate nducton wth low-dose G suggests that drug nteractons should also be expected wth ths therapeutc regmen. 2) lteratons n drug metabolsm mght be expected f a patent s transferred to another G drug schedule.

5 18l 3) The nonselectve nducton of metabolsm of both warfarn somers shows that drug nteracton between G and warfarn cannot be avoded by admnstraton of one somer. 4) Several sterods, ncludng estrogens, are metabolzed by hydroxylatons catalyzed by the mxed functon oxdase system. The possble role of a dose-dependent G-promoted nducton of sterod metabolsm n the mechansm of acton of ths drug, s a subject under study n our laboratory. References 1. mercan College of Clncal Pharmacology (1982) Manual of symbols, equatons and defntons n pharmacoknetcs. J Cln Pharmacol 22: ls 2. Breckenrdge, Orme HL, aves L, Thorgersson S, aves S (1973) ose-dependent enzyme nducton. Cln Pharmacol Ther 14: owsett M, Jeffcoate SL, Santner S, Santen RJ, Stuart-Harrs R, Smth E (1985) Low-dose amnoglutethmde and aromatase nhbtors. Lancet 1 : Harrs L, owsett M, Smth IE, Jeffcate SL (1983) Endocrne effects of low dose amnoglutethmde alone n advanced postmenopausal breast cancer. Br J Cancer 47: 62l 5. Harrs L, Cantwell BMJ, Sansbury JR, Needham G, Evans RGB, awes PJK, Wlson R, Farndon J (1986) Clncal effects of low-dose amnoglutethmde (G) plus hydrocortsone (HC) n advanced breast cancer. Breast Cancer Res Treat (n press) 6. Hewck S, McEwen J (1973) Plasma half-lves, plasma metaboltes and antcoagulant effcaces of the enantomers of warfarn n man. J Pharm Pharmacol 25: Kelly JG, O'Malley K (1979) Clncal pharmacoknetcs of oral antcoagulants. Cln Pharmacoknet 4:1 8. Kvnnsland S, Lonnng PE, ahl O (1984) Treatment of breast carcnoma wth amnoglutethmde. cta Radol Oncol 23: Lews R J, Trager WF, Chan KK, Breckenrdge, Orme M, Roland M, Schaby W (1974) Warfarn. Stereochemcal aspects of ts metabolsm and the nteracton wth phenylbutazone. J Cln Invest 53: Lonnng PE, Kvnnsland S, Bakke OM (1984a) Effect of amnoglutethmde on antpyrne, theophyllne, and dgtoxn dsposton n breast cancer. Cln Pharmacol Ther 36: Lonnng PE, Kvnnsland S, Jahren G (1984b) mnoglutethmde and warfarn. new mportant drug nteracton. Cancer Chemother Pharmacol 12: Lonnng PE, Schanche JS, Kvnnsland S, Ueland PM (1985) Sngle-dose and steady-state pharmacoknetcs of amnoglutethmde. Cln Pharmacoknet 10: Murray FT, Santner S, Samojlk E, Santen RJ (1979) Serum amnoglutethmde levels: Studes of serum half-lfe, clearance and patent complance. J Cln Pharmacol 19: Murray RML, Ptt P (1985) Low-dose amnoglutethmde wthout sterod replacement n the treatment of postmenopausal women wth advanced breast cancer. Eur J Cancer Cln Oncol 21 : Ohnhaus EE, Park BK (1979) Measurement of urnary 6-betahydroxycortsol excreton as an n vvo parameter n the clncal assessment of the mcrosomal enzyme-nducng capacty of antpyrne, phenobarbtone and rfampcn. Eur J Cln Pharmacol 15: O'Relly R (1974) Interacton of sodum warfarn and rfampn. nn Intern Med 81 : O'Relly R (1976) The steroselectve nteracton of warfarn and metrondazole n man. N Engl J Med 295: O'Relly R, ggeler PM, Leong LS (1963) Studes on the coumarn antcoagulant drugs. The pharmacodynamcs of warfarn n man. J Cln Invest 42: O'Relly R, Wellng PG, Wagner JG (1971) Pharmacoknetcs of warfarn followng ntravenous admnstraton to man. Thromb ath Haemorrh 25: Park BK, Breckenrdge M (1981) Clncal mplcatons of enzyme nducton and enzyme nhbton. Cln Pharmacoknet 6: l 21. Santen RJ, Lpton, Kendall J (1974) Successful medcal adrenalectomy wth amnoglutethmde. Role of altered drug metabolsm. JM 230: Santen RJ0 Santner S, avs B, Veldhus J, Samojlk E, Ruby E (1978) mnoglutethmde nhbts extraglandular estrogen producton n postmenopausal women wth breast carcnoma. J Cln Endocrnol Metab 47: Schanche JS, Lonnng PE, Ueland PM, Kvnnsland S (1984) etermnaton of amnoglutethmde and N-acetylamnoglutethmde n humanplasma by reversed-phase lqud chromatography. Ther rug Mont 6: Stuart-Harrs R, owsett M, Bozek T, McKnna J, Gazer JC, Jeffcoate SL, Kurkura, Carr L, Smth IE (1984) Lowdose amnoglutethmde n treatment of advanced breast cancer. Lancet 2: Ueland PM, Kvalhem G, Lonnng PE, Kvnnsland S (1985) etermnaton of warfarn n human plasma by hgh-performance lqud chromatography and photodode array detector. Ther rug Mont 7: Vermeulen, Pardaens R, Heuson JC (1983) Effects of amnoglutethmde on adrenal sterod secreton. Cln Endocrnol 19:673 Receved July 4, 1985/ccepted ecember 5, 1985