[CANCER RESEARCH 55, 810—816,February 15. 19951

cyclophosphamide have not been previously modeled. Explicit modcling of the disposition of cyclophosphamidepermits a formalexplanationof thesephenomena.

We have carried out a comprehensive pharmacokinetic analysis ofcyclophosphamidein 15patientswith metastaticbreastcancerundergoing high-dosechemotherapywith alkylating agents followed byautologousbonemarrowtransplantation.There areseveralreasonsforconducting the current investigation. (a) Some aspectsof cyclophosphamide dispositionoccurringat high dose may not appear at theconventionallow doses,e.g., a saturableeliminationprocess(8). (b)Previousand/or concomitantadministrationof microsomalenzymeinducing agents (phenobarbital and phenytoin) or enzyme-inhibitingagents(morphineandprogesterone)with cyclophosphamidemay altercyclophosphamidekinetics and thereby its therapeuticand toxic responses(2, 13, 17, 18). (c) Several clinical studies have observed theincreasedclearanceanddecreasedt½andAUC@of cyclophosphamideafter repeatedtreatments indicating that cyclophosphamide induces itsown metabolism(autoinduction)(12, 14—16,19). Finally, becausepolychemotherapyis frequently administered,the inducing capacityof cyclophosphamidedeservesfurther investigation,sincecyclophosphamide could not only induce its own metabolism but also themetabolismof otherdrugsadministeredsimultaneously(heteroinduction; Refs. 18 and 19).

We have applied pharmacokinetic models that describe (a) thedose-dependentkineticsof cyclophosphamidewhen administeredinhigh dose by a short infusion and (b) the time-dependent kinetics ofcyclophosphamidewhen given by a prolongedinfusion. The dosedependentkineticmodelconsistsof a saturableeliminationprocessinparallel with first-order renal elimination of cyclophosphamide. Thetime-dependent kinetic model consists of a gradual change in cyclophosphamide clearance following a latency period. The clinical responses and toxicities of this treatment will be the subject of aseparatereport.

PATIENTS AND METHODS

Patient Population and Study Design. Women with stage IIIB or IVbreastcancerundergoingautologousbone marrow transplantationwere eligible for thisstudy.Patientswererequiredto havehistologicallydocumentedbreastcancerresponsiveto conventionaldosesystemicchemotherapy,to bebetween18and60yearsold, to haveEasternCooperativeOncologyGroupperformance status less than 2, normal hematopoietic function, and adequatecardiac (left ventricle ejection fraction, >45%), pulmonary (forced vital capacity and forced expiratory volume in 1 s, >60% of predicted for patient's heightandweight),renal(serumcreatinineconcentration,<2.0 mg/dl),andhepatic(serumAST concentration,<60lU/mi andserumbilirubin concentration,<1.5mg/dl) functions. The study was approved by the Joint Committee for ClinicalInvestigation of the Johns Hopkins Hospital and written informed consent wasobtained from each patient.

After the bone marrow was harvested, the patients received a first course of

cyclophosphamide (4 g/m2 administered i.v. over 90 mm) for mobilization ofperipheralblood progenitorcells.Threeweekslater, the patientsreceiveda

3 The abbreviations used are: AUC, area under the curve; CYP, cytochrome P450.

810

Nonlinear Pharmacokinetics of Cyclophosphamide in Patients with Metastatic

Breast Cancer Receiving High-Dose Chemotherapy followed by

Autologous Bone Marrow Transplantation'

Tian-Ling Chen,2 Jose L. Passos-Coelho, Dennis A. Noe, M John Kennedy, Kraig C. Black, 0. Michael Colvin,and Louise B. GrochowThe JohnsHopkins OncologyCenter, Division of Phannacologyand ExperimentalTherapeutics,Baltimore, Maryland 21287

ABSTRACT

The pharmacokineticsof cyclophosphamidehas beenevaluatedin 15patients with metastatic breast cancer undergoing high-dose chemother.apy with alkylatingagentsfoflowedby autologousbonemarrow trnn@.plantation. Each patient received two courses of chemotherapy: 4 Wan2ofcyclophosphamide by 90-miss infusion prior to peripheral blood progenitar cellcollection(thefirst course)and6 g/m2of cyclophosphamidewith800 mg/rn2 of thiotepa by 96-h constant infusion before marrow and stemcell reinjection (the second course). In the first course, plasma cyclophosphamide concentration-time data of 9 of 15 patients were fit by a onecompartment model with Mlchaelis-Menten saturable elimination in par.allelwithfirst-orderrenalelimination.Themean(SD)Vm,,,,andKmvalueswere 1.47 (0.89) paM/mmand 575 (347) pM, respectively. The first coursedata of the remaining six patients were fit using first-order eliminationonly. In the seconddrug course, plasma cyclophosphamidedispositioncurves of 13 of 15 patIents demonstrated a decline In concentration

followingattainmentof an Initial steadystate.The plasmacyclophosphamide disposition data of these patients were fit by a one-compartmentpharmacokineticmodel,in which thedeclineofplasma cyclophosphamideconcentration after reaching the Initial steady state was modeled as beingdueto an increasein the clearancerate of cyclophosphamide.The mean(SD) Initial and final clearancerateswere51 (16) mI/missand 106 (48)mi/win, respectively.Mlchaelis-Mentenelimination was not apparent inthesecondcoursebecausetheplasmaconcentrationof cyclophosphamidewas much lower. The mean renal clearance rate was 17 mI/mm in the firstcourseand 16 mI/mm in the secondcourse.Urinary excretion of cyclephosphamide accounted for 17% and 23% of the total dose administeredin the first and the secondcourse,respectively.No changein cyclophos.phamide clearance rate was apparent In a patient who was taldng phe.nytoin, but a changewaspresentin a patient who wastaking phenobarbital. A drug interaction between cyclophosphamide and thiotepa mayexplain the smaller Initial clearancerate for cyclophosphamideduring theseconddrug course.

INTRODUCTION

High-dose chemotherapy with alkylating agents followed by autologous hematopoietic rescue hasbeen shown to increase responserateand overall survival in selected patients with advancedcancers (1, 2).Cyclophosphamide is one of the cytotoxic drugs most commonly usedin high-dose chemotherapy prior to bone marrow transplantation. It isa prodrug that undergoes complex metabolic activation and detoxification. The chemistry, pharmacology, and pharmacokinetics of cyclophosphamide have been extensively studied (reviewed in Refs. 3—10).Several investigators have reported on the disposition of high-dosecyclophosphamide in patients (2, 11—16),noting apparent dose- andtime-dependent kinetics. However, the nonlinear kinetics of high-dose

Received9/15/94;accepted12/15/94.The costsof publicationof this articleweredefrayedin partby thepaymentof page

charges.This articlemustthereforebe herebymarkedadvertisementin accordancewith18 U.S.C. Section 1734 solely to indicatethis fact.

I This study was supported in part by Grants CA15396 and CA63437 from NIH andby Maryland Medical Laboratory, Inc. M. J. K. is a recipient of an American CancerSocietyClinical OncologyCareerDevelopmentAward.

2 To whom requests for reprints should be addressed, at The Johns Hopkins OncologyCenter,Room1—121,600 North Wolfe Street,Baltimore,MD 21287.

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PHARMACOKINETIcSOF CYCLOPHOSPHAMIDEIN PATIENTS

combination of cyclophosphamide (6 g/m2) and thiotepa (800 mg/m2) administeredsimultaneouslyas a 96-h continuousi.v. infusion. Novobiocin (2 gevery 12 h orally for 14 doses starting 36 h prior to the chemotherapy) wasaddedto inhibit the developmentof alkylating agentresistance(20). Ondansetron (0.15 mg/kg loading dose followed by 1 mg/mm continuous i.v. infusion) and lorazepam (1 mg every 4 h iv.) were administered from the timechemotherapyinfusion starteduntil 24 h after treatmentfinished.Prochlorperazine(5 mg every3 h i.v.) wasgiven asneeded.

Bloodandurinespecimenswerecollectedfor thedeterminationof plasmaand urine cyclophosphamideconcentrationsduring both coursesof therapy.For the first course(90-mminfusion),blood sampleswereobtainedat 0, 45,and80mm,andat 2, 3, 4, 5, 8, 10,16,and24 h measuredfrom thebeginningof the infusion. For the secondcourse(96-h infusion),blood sampleswereobtained at 0, 3, and 6 h, then every 6 h during the cyclophosphamide infusion,andat 3 and6 h aftertheendof theinfusion.Urinewascollectedup to 36and120 h during the first and the second coursesof treatment, respectively.Separated plasma and urine aliquots were stored at —20°Cuntil analysis.

Analytical Methods. Plasma cyclophosphamide levels were measured bygas chromatography using a modified method based on those of Jardine a aL(21), Juma et aL (22) and Dr. B. Gourmet.4 A Varian 3400 gas chromatograph(Varian, Sugar Land, 1'X) equipped with a thermionic specific detector wasused. The separation was achieved isothermally on a 1.82 m x 2 mm insidediameter glass coil packed with 3% SP 2250 on 80/100 mesh Supelcoport(Supelco, Bellefonte, PA). The gas chromatography operating conditions wereasfollows: theinjector,column,anddetectortemperatureswere220,220,and300°C,respectively; nitrogen was used as the carrier gas at a flow rate of 25ml/min; hydrogen and air flow rates for the thermionic specific detector were4 and 240 ml/min, respectively. A Dell 486D/25 personal computer (Dell,Austin, TX) and PE Nelson2600chromatographicsoftware(Perkin Elmer/Cettis, Morrisville, NC) were utilized for the data acquisition, calibration, andconcentrationcalculation.The samplepreparation,briefly described,is asfollows: to each 0.5 ml of plasma(1 ml of urine plus 100@ of 1 N NaOH),30 @.dof 1 mM ifosfamide as an internal standardand 5 ml of ethyl acetate(methylenecholoride for urine) were added.After shakingfor 10 mm, themixture was centrifuged at 2000 X g for 10 mm. The supernatant wastransferred to a clean tube and evaporated to dryness under a stream of N2. Theresiduewas derivatizedwith 100@ ethyl acetateand50 pi heptafluorobutyricanhydride (Sigma, St. Louis, MO) and incubated at 70°Cfor 2 h. AfterevaporatingunderN2,theresiduewasreconstitutedwith 1 ml of ethyl acetate.Samples were injected automatically using a Varian 3400 autosampler. Theretentiontimes for ifosfamideandcyclophosphamidewere 2.6 and3.4 mm,respectively. The lower limit of detection of cyclophosphamidewas 1 p@M.Heptafluorobutyricanhydridewasusedinsteadof trifluoroaceticanhydridetomaximize the sensitivity and to enable reconstitution of the residue in a volumewhich would be suitable for the autosampler.

Plasmaphenytoinandphenobarbitallevelswere measuredby fluorescentpolarization immunoassayscourtesyof Maryland Medical Laboratory,Inc.(Baltimore,MD).

Pharmacokinetic Modeling. Plasma cyclophosphamide disposition curveswere first examinedvisually to assesssuitable models.After the 90-mminfusion,many patientsexhibitedconvex-downwardelimination curves,whichis typical of a Michaelis-Menten saturable elimination process (23). We testedthe fit of theplasmacyclophosphamidedispositiondatafrom the first courseby a one-compartment model with Michaelis-Menton saturable eliminationcoexisting with first-order renal elimination (23):

dC R ,@, V@,C@ - V- —V----@ -

wheredC/dt is therateof changeof drugconcentrationat time :, R is therateof infusion (t > infusion time, r = 0), V is the apparent volume of distribution,Cirena! is the renal drug clearance rate, Vm,,, is the theoretical maximum rate of

theeliminationprocess,andKmis theMichaelisconstant.A weightof 1/Cwasusedin the iterativefitting process.Renalclearance,Clr€,...@,wasobtainedasfollows:

xCl renal

whereX@is the amountof cyclophosphamideexcretedin the urine andAUCis the extrapolated area under plasma cyclophosphamide disposition curvecalculatedusinga combinedlinearandlogarithmictrapezoidalrule (24). Forthis model, the systemic clearance rate varies with concentration and wascalculated as follows (23):

vvCl =@ +C1@,

Maximum plasma concentrations of cyclophosphamide during the 96-h infusion were 20% of the Cmi,, after 90-mm infusion, therefore, a MichaelisMentensaturableeliminationprocesswas not observed.However,decliningplasmacyclophosphamideconcentrationswereapparent.Oneway to accountfor this is to modelclearanceas increasingover time. Severalmodelsof thisincreasewere evaluated,including a step function increasein clearance,asigmoidal increase in clearance, and an exponential increase in clearance(25—27).The model that fit the data best was that with an exponential increasein clearance, in which the full pharmacokinetic model was:

and

dC R Cl(t)-@=V--—-@--C (Model2)

C1(t)=@ —(Cifinal CI1@1@,)e@'―@for t > tiag;

where Cljnitjaj ts the initial clearance rate and@ is the final clearance rate,4, is the rate constant for the change in clearance rate, and@ @5the latencyperiod, i.e., the time from the commencement of the drug infusion to thebeginining of the increase in clearance.

For datasetsthatwere not well fit by the abovemodels,i.e., in the first drugcourse,when the asymptomaticstandarderror for Kmwas >30% associatedwith estimates of Km > 2Cpiasma.or in the second drug course, when visualinspectionshowedno changesin clearance,Model 1 or Model 2 collapsedtoModel 3, a one-compartmentmodelwith constantinput andfirst-orderdimination (23):

dC R-@=V-KC (Model3)

where K is the rate constantfor the first-orderelimination. Cm,,,and Csswerebasedon modelfitting, andnoweightingfactorwasusedfor the96-hinfusioncourse.

All pharmacokinetic modeling and parameter estimates were performed bynonlinearregressionanalysisusing PCNONLIN (Statistical Consultants,Lexington, KY). The code for Model 1 and Model 2 can be obtained from theauthors of this report, since they are not available in the PCNONLIN library.The mean±SD of pharmacokineticparameterswascalculatedusingQuattroPro for Windows(BorlandInternational,Inc., ScottsValley, CA).

RESULTS

cyclophosphamide disposition curves and urinary excretion data forboth coursesof treatment.

For the first course,each patient received 4 g/m2 of cyclophosphamide over 90 mm. Plasma cyclophosphamide disposition curvesfrom 9 of 15 patientswerewell fit by Model 1 with Michaelis-Mentensaturable elimination and first-order renal elimination in parallel. Theelimination parameter estimates are given in Table 1. The asymptomatic standard errors of the parameter estimates were less than 15% ofthe estimates.When Cpi,@ma@5lessthan or in the vicinity of Km, thepostinfusion profile demonstrated a convex curve (Fig. 1A); when

Cl(:) = Cl@,, for 0 t@

Fifteen of 20 women with stage IIIB (1 patient) or IV (14 patients)breastcancerundergoingautologousbone marrowtransplantationbetweenApril 1993 andOctober 1993 hadpharmacokineticsamplingperformed and are included in this report. The median age of the

(Model 1) patients was 44 (range, 31—60) years. Two patients had liver metas

tasisbut normal liver enzymes.All 15 patientshad completeplasma

4 Personal communication.

811

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Table 1Cmax and dimsnationparametersfor nonlinearfitring (firstcourse)PatientCmax

(PM)Vm,,,, (@min)Km (MM)a@na@/v(min@)2

346789

1213499

5726656005135436015074701.28

2.851.081.860.810.713.090.361.18537

924237974352367

11991424420.0004

0.00010.00040.00060.00050.00040.00030.0007

0.0004Mean

SD552 591.47 0.89575 3470.0004 0.0002

PHARMACOK1NFI1@SOFCYCLOPHOSPHAMIDEIN PATIENTS

parametersfor the first course.The overall volume of distributionremained unchanged in the first and the second drug courses (range,26—55liters; patient 1 had an outlying value for the volume, 153liters, in the secondcourse).

Patients1 and 14 were takingphenytoinandphenobarbital,respectively, before and during both coursesof treatmentfor preexistingseizure disorders. Interestingly, patient 1 had a very high clearancerate in the first course;shehad the lowestCss(45 p@M)in the secondcourse and demonstratedno decline in plasma cyclophosphamidelevels after reaching the steady state (Fig. 3). Patient 14 had a

500

@200

@100jso

.@ 20

.@ 10

2

400 800‘rime(mm)

ACptasma @5much greater than Km, the pseudolinear portion is apparent

(Fig. 1B).The dispostion curves of the remaining six patients did not appear

to be convex, possibly because the elimination Km5 in these subjects

were much larger than the plasma cyclophosphamide concentrationsachieved. When Km values are much greater than the plasma drugconcentrations, Model 1 reduces to

dC R Cl,,,@ V@,V,,,,,C R fCl@ V,@ V,@\ Rdt V V C v K,, V@ V@ V@ Km ) C V K@ C

where Ktotai@5a first-orderrate constantof eliminationconsistingofthe first-order renal elimination rate constant, Clre,.,@i/V,and the (jseudo) first-order nonrenal elimination rate constant, Vmas/Km.The cyclophosphamide plasma disposition curves of these patients were fitby Model 3 (Table 2). Fig. 1C showsa typical plasmacyclophosphamide concentration-time profile for one such patient.

For the secondcourse,eachpatientreceived6 g/m2 of cyclophosphamide over 96 h. Plasma cyclophosphamide disposition curves of

13 of 15 patients demonstrated a constant decline in “steady-state―levels of cyclophosphamide during the infusion after having reachedan initial steady-statevalue, Cssjnifi@(Table 3). The plasmacyclophosphamideconcentration-timedatafrom ninepatientswere well fitby Model 2. The estimates of 4 and tlagare listed in Table 3. The 4ivalue represents the rate constant of the increase in clearance rate.Simulation (not presented) shows that when 4 is very small (<0.0001min 1),estimatesof 4 are uncertainbecausethe infusiontime is tooshort to approach the final steady state; when 4 is very large (@1min ‘),the 6-h sampling interval doesnot permit reliable estimatesof4@.Theincreaseinclearancerateofcyclophaphamidestarted,anaverage,29 h after the infusionbegan.Fig. 2 showsa typical plasmacyclophosphamide concentration-time fit by Model 2.

The useof Model 2 to fit the data for the other four patients resultedin very large asymptomatic errors of at least two parameter estimates.

For example, the data of two patientscould be fit equally well by a4,= 1,0.1,or0.01min'.

Plasmacyclophosphamideconcentrationsfor patient 13 were chaotic (showed no consistent behavior), so thesedata were fit by Model3 instead of Model 2. It is unclear whether this was due to unusualphysiological conditions or to the failure of adequatesampling (i.e.,venous accessdifficulty, etc.).

There was interpatient variability in the urinary excretion andclearance of cyclophosphamide (Table 4). Urinary excretion of unchanged cyclophosphamide ranged from 4 to 38% of the total doseadministered. The renal clearancerate was approximately the sameforboth courses (17% and 16%, repectively). For the purpose of comparing the clearancerate estimatesfrom the first and seconddrugcourses, the systemic clearance rate in the first course was calculatedat the C55initiaiand in the second course for patients whose data hadbeen fit using Model 1. The measured Cl1@1@1@in the second course

was 56% lower than the expected clearance rate calculated from

II

Time (mm)

B

1,200 1,600

C

Time (mm)

IFig. 1. Time courseof plasmacyclophosphamideconcentrationsduring 90-min infu

sion at a doseof 4 g/m2for: (A) patient4, V,,,.,, 108 @u.i/min,Km 237 p.@i;(B)patient9, V@,j, 309 g@.M/min,Km 1199 ,LM;(C) patient 14 with linear fitting,K = 0.0031. Lines, model fit.

812

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Table2 C,,,,@andeliminationparameterfor linearfitting (firstcourse)PatientCm,.,.

(I@M)K(fl1ifl@')15870.009856220.0025106210.0021116120.0031144820.0031157000.0033Mean6040.0040SD650.0026

Table3 Cssandpharmacokineticftparameters

for time variantflrst-ortting (second course)der

eliminationPatientCssjflj@i.i

(pM)(pM)4, (min')tiag(m1n)27553310777411574S119910.00101724676560.000121967120810.0005154581431140.000125009118830.000118861015213411119770.0006109912109880.004416201493670.0003112215124940.00182027Mean113840.00101747SD21210.0013439

@u@t@coiur-miicsOFCYCLOPHOSPHAMIDEIN PATIENTS

evidencedin the absenceof detectablenonlinearity in the clearancecurves of the remaining six patients, presumably due to the fact thateffectiveKmvaluesfor theseindividualswere higherthanthe plasmaconcentrationsachieved. Nonlinear elimination exhibited by somepatientstreatedwith high dosesof cyclophosphamidemay increasethe risk of potentially lethal toxicities; unmetabolized cyclophosphamide will not be completelyexcretedunchangedin urine andwill beavailable longer for metabolism to cytotoxic forms.

During a 96-h constant infusion, cyclophosphamide concentrationsdeclined after achieving an initial Css, suggesting time-dependentelimination kinetics due to enzyme induction (23). Many drugs inducetheir own metabolism (autoinduction) as well as that of other drugsadministered concomitantly (heteroinduction). The autoinduction ofcyclophosphamide metabolism in humans was first proposed by Bagley et a!. (13) in 1973. They found that at dosesbetween 6 and 80mg/kg,['4C]-cyclophosphamide stimulated its own metabolism withinthe first 2 days of consecutive daily administration and then continuedto be metabolized at the higher rate until at least the fifth day oftreatment.The t@of cyclophosphamidewas shorterand the effectivealkylating metabolites were consistently higher on the fifth day thanon the first day of drug administration. Other investigatorshavereported a time-dependent decline in t@ and AUC after sustainedtreatmentwith cyclophosphamide(11—16,19). In contrast,Mouridsenet al. (29) found no change in the t@ after 22 days of treatment with

Fig. 2. Time courseof plasmacyclophosphamideconcentrationsduring 96-h infusionat a doseof 6 g/m2 for patient 11.

Fig. 3. Time courseof plasmacyclophosphamideconcentrationsduring96-h infusionin thepatientwho wastakingphenytoin(patient1,A) andphenobarbital(patient14,U).

dispositioncurve that did not differ from the other 13 patientsin thesecond course (Fig. 3). The data of patient 1 were fit by Model 3 andthoseof patient 14 were fit by Model 2.

Phenytoin and phenobarbital levels were measured in all plasmasamples for patients 1 and 14, respectively (Fig. 4). Phenytoin levelsin the second course from patient 1 were higher than in the firstcourse,andremainedunchangedduringthe secondcourse.Phenobarbital levels from patient 14 startedhigher and declined during thesecond course.

DISCUSSION

Cyclophosphamide is a prodrug requiring hepatic microsomal en

zyme activation to its active metabolites. Several CYP proteins havebeenidentified asthe enzymescapableof metabolizingcyclophosphamide in humans (28). Because the biotransformation of cyclophosphamideis enzymecatalyzed,at cyclophosphamideconcentrationsinthe vicinity of and greaterthan Km, nonlinearelimination kineticsofthe drugshouldoccur(8). Studieshavenot shownnonlinearbehaviorin patientsreceivingcyclophosphamideat low doses.Mouridsenet al.(29) studiedthedosedependencyofcyclophosphamideandfoundthatthe serum t@of cyclophosphamide,the serumconcentrationof metabolites, and the rates of excretion of cyclophosphamide and metaboliteswere independentof doseover the range0.02—10mg/kg. Brocket aL (30) reported that the rate of cyclophosphamide activation wasindependent of dose over the range 1—2g/m2. The possibility ofsaturable kinetics in patients receiving high doses of cyclophosphamide for bone marrow transplantationwas predictedto occur whenplasma concentration reached 500—600p.M (8). We have identifiedsaturable elimination of cyclophosphamide in 9 of 15 patients treatedwith 4 g/m2 administered by 90-min infusion. In these subjects, themean Cm@, S'max,and Km were 552 p.M, 1.47 p@M/min,and 575 p.M,respectively. Since several enzymes (with low and high K,,,s) inhuman liver are catalytically capable of metabolizing cyclophosphamide (28), the Km estimated may represent the metabolic capacity ofa groupof enzymes.Interindividualvariability in theeffectiveKmwas

4,000

Time (mm)8,000

150

813

2,000 4,000

Time (mm)

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Table4 Urinary excretionand ciearancerates ofcyclophosphamideUrinary

excretion (% of dose)Q@ (mi/min)

@ IICI,@ystemk

(mWn1in)

I@ @Patientr

11b1

2c3C

4C

56c7C8'@

9C101112c13c14157

1020 244 10@5 17

20 2324 2723 35

24 3012 23

15 2412 1033 3815 2721 2913 1920

1519 235 8

13 1418 1226 2925 2519 1511 1411 1012 628 2416 1725 2112 11296

15310813712390 40101 95111 5078 4494 5173101 3889 48117 62120 5397 45153

642179999162

83696297

65Meand

SD―1723

7 81716

7 711651

51 16106 48

PHARMACOMNEfl@5OFCYCLOPHOSPHAMIDEIN PATIENTS

100% higher than the initial clearance rate. The change in clearancerateof cyclophosphamidewasconsistentwith that reportedby Mooreet al. (11). They found that the mean clearance rate in patientsreceiving 50—60mg/kg daily for 2 consecutive days were 93 and 178mi/mm for days 1 and 2, respectively. In this study, the volume ofdistributionwas the sameduring the first and the secondcoursesoftreatment.Although Graham et aL (14) reportedthe same result, adecreasedvolume of distribution was noted in patients after continualcyclophosphamidetreatmentat low doses(19). On the basisof previously publishedstudiesof single-agentcyclophosphamideautoinduction, we believe that the decline in plasma cyclophosphamideconcentrationduring a prolonged infusion is due to induction ofhepatic microsomalenzymesby cyclophosphamide.This inductionappearsto occur at a mean of 29 h after the infusion begins, comparable to that previouslyreportedin both adultsand pediatricpopulationswhen cyclophosphamidewas given daily for 2 to 4 consecutivedays(11, 15) andalsocomparableto estimatesof CYP autoinductionby other agents(25, 31, 32).

Cyclophosphamide disposition hasbeen described with biexponential modelsafter rapid i.v. injection(7, 33). The initial t@, was 6—13min (22, 33). Sincea “distribution―phasemay not be evidentduringa prolongedinfusion,a one-compartmentmodel is utilized to describethe kinetic behavior of cyclophosphamidefor both courses oftreatmentin this study.

The coadministrationof cyclophosphamidewith novobiocin andthiotepain the secondtreatmentcourseappearedto affect the clearance rate of cyclophosphamide. The renal clearance rate was nearlyidentical in the two coursesof treatment. However, the initial nonrenalclearance rate in the second course was considerably lower thananticipatedbased on estimatesfrom the first course.With a 96-h

B

a@ course I; II, course II. 4 g/m2/90-min infusion.6 g/m2/96-h infusion.

C Cl@ystemic for course I is calculated at plasma Cssm@,@ obtained in course II (see

Table3).d Patients 1 and 13 are excluded in calculations for Cl@y.tcmic for course U.

2 mg/kg of cyclophosphamide. Ayash et a!. (2) reported cyclophosphamide dispositionat 6 g/m2/96-h infusion and demonstratedthesame decline in steady-state concentrations as are described in thisarticle. However, these authors did not attempt to model the timedependency.In our study, plasma dispositioncurves of 13 of 15patients demonstrated such declines. After the concentration reachedthe Css.njtiai,it steadily decreasedto a mean 30% lower than thatvalue. At 96 h (the end of infusion), the apparent clearance rate was

A

•• •. .

S@ .

C. •

14

a.oos 4.O@ •.one 1.4@me

D

14

I@1o

Fig. 4. Time course of plasma phenytoin (•)andphenobarbital(U)concentrationsfor:(A)phenytoinconcentrationsin first course;(B) phenytoinconcentrationsin secondcourse;(C) phenobarbitalconcentrationsin first course;(D) phenobarbitalconcentrationsin secondcourse.

UU

U UU U

@ UU@

U U U U U

12

I

Cs •S

S • S S

IS

0 300@@@ 1.000 1.@O 1.400 IA

@ms(ni@

C

11

@:. .. C@uo—. U

1'I'

a

_o@@ 1.a@ 1.3@ 1.4w lAOS@ms@

_o@ LOOS 4.OOSThOS

814

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PHARMACOKINETI@@SOF CYCLOPHOSPHAMIDEIN PATIENTS

continuous infusion, the percentageof the drug excreted unchanged inurine (i.e., never metabolized to the active compound) was higher,since the metabolic clearancewas smaller and more drug was excretedunchanged. There are several factors that may have reduced thenonrenal clearance rate, including the physiological condition andhydration status of the patients, altered protein binding, and druginteractions. All patients had minimal metastatic breast cancer andtheir overall physiological condition was excellent. There were nomajor physiologicalchangesbetween the two coursesof treatment.All patients received intensive peripheral hydration during bothcourses to minimize bladder toxicity. Since the@ was about thesame for both courses, it is unlikely that hydration status alteredcyclophosphamide clearance. Although protein binding (e.g., to anacute phase reactant such as a@acid glycoprotein) could be altered,the modest protein binding of cyclophosphamide (12—24%;Ref. 7) isunlikely to alter drug clearance. Thus, drug interactions may beimplicated. Cyclophosphamide was coadministered with novobiocinand thiotepa during the second course, whereas it was given as asingle agent during the first course. A Phase I study has shown thatnovobiocin at doses between 0.5 and 5 g/day did not affect cyclophosphamide concentration.5 Thiotepa is largely metabolized totepa by a saturable hepatic enzymatic process (34, 35). The CYPenzyme for metabolizing thiotepa to tepa has not yet been definedin humans. However, in the rat system, the CYP enzymes responsible for thiotepa activation (PB1, 2c, and PB-4) were the same asthose for cyclophosphamide (36, 37). It is possible that thiotepacompetes with cyclophosphamide for liver CYP metabolism resulting in altered metabolic clearance. This possibility should beinvestigated further in vitro in an isolated CYP system (28, 37—39)and in vivo by administration of cyclophosphamideand thiotepaconsecutively rather than concurrently.

Autoinduction of cyclophosphamide clearance was not demonstrated in the patient who was receiving phenytoin; she had thehighest clearance rate in the first course and the lowest Css(associated with an atypical large volume of distribution) in thesecond course. Sladek et a!. (16) reported that additional inductionwas not seen in a patient who was taking phenytoin before andduring cyclophosphamidetreatment. Phenytoin is metabolized to4-hydroxyphenytoin by the hepatic microsomal enzyme CYP2C9/10 (39). Although the CYP 2B subfamily preferentialy catalyzes cyclophosphamide hydroxylation, CYP 2C9 is also catalytically competent (28). It is possible that cyclophosphamide metabolism may be maximally induced in patients receiving phenytoin,in whom further induction will not occur. Plasma phenytoin concentrations in the second course in this patient were higher thanduring the first course, suggesting a decreased phenytoin clearancerate during concomitant infusion of cyclophosphamide and thiotepa. There was no decline in phenytoin concentration over the96-h infusion, indicating that the induction of cyclophosphamideclearance was not also affecting phenytoin clearance. The autoinduction of cyclophosphamide metabolism was clearly demonstrated in patient 14, who was receiving phenobarbital. Althoughconflicting results have been reported regarding the decrease incyclophosphamidet½in patients pretreatedwith phenobarbital (7,16), our observationis in agreementwith that reportedby Jaoet a!.(40). They found that phenobarbital had little effect on the metabolism of cyclophosphamide in patients who were preinduced byphenobarbital. Plasma phenobarbital concentrationsin this patientwere higher at the beginning of the secondcourseand continuouslydeclined during 96-h constant infusion. The CYP enzyme respon

sible for metabolizing phenobarbital in humans has not been identified. It is not clear whether the alteration of phenobarbital clearance was due to cyclophosphamide and/or thiotepa. Ng andWaxman (37) reported that the metabolism of thiotepa to tepa wasacceleratedin rats preinduced by phenobarbital. If this is the casein humans, the decreasedphenobarbital levels may be caused bythiotepa heteroinduction.

The fmdings of this study have significant clinical implications. (a)The saturableelimination of cyclophosphamideafter a short druginfusion at high doses suggests that when the dosing rate equals orexceeds4 g/m2/90 mm or the plasmaconcentrationof cyclophosphamide exceeds 150 p.M (the lowest K,,j, nonlinear elimination mayoccur, resulting in altered disposition curves for both cyclophosphamide and its active metabolites.Variations in the nonrenalclearancerate may also produce relative underexposure to the principal cytotoxic metabolitesfor patientswho excretea large fractionof the doseunchanged in urine. We are currently investigating the possibility ofalterations in drug effect associatedwith such altered drug disposition.Our study indicatesthat nonlinear elimination is unlikely to occurduring a prolongedinfusionat a dosingrate of 6 g/m2/96 h. (b) Theinduction of cyclophosphamide clearance leading to a decrease incyclophosphamide concentrations during a prolonged infusion meansthat blood sampling must be undertaken througout the infusion toassesscyclophosphamide exposure during such therapy. (c) In theseconddrugcourse,the smaller initial clearancerate of cyclophosphamide we report may only be pertinent to the coadministration ofcyclophosphamide and thiotepa used clinically as a preparative regimen in bone marrow transplantation. (d) Cyclophosphamide andthiotepaand/ortheir metabolitesmay inhibit the clearanceof phenytoin andenhancethe clearanceof phenobarbital.This hasimplicationsregarding seizure control and antiseizure drug toxicity during highdose cyclophosphamide therapy.

Drug metabolismand enzyme inductionoften exhibit large interindividualvariability. The variability in the percentageof parentdrugexcretedin urine,thecapacityof themetabolicprocess,andtheextentof induction in a subject for a given drug is not predictable. Althoughsome authors have suggested the use of inducing agents in cancerpatients to enhance drug activation, this approach may be hazardous,producing more frequent lethal toxicities. It would be more rationaland practical to give a single dose and assess drug and metabolite

exposure and relevant toxicity.We have found nonlinearnonrenalelimination of drug in 9 of 15

patients receiving short cyclophosphamide infusion at high doses,andinduction of nonrenal cyclophosphamide clearance in 13 of 15 patients receiving drug by a 96-h infusion. In addition, we have identifled a potential drug interaction of cyclophosphamide with thiotepaleading to decreasednonrenal clearance, and possible interactions ofcyclophosphamidewith phenytoinand phenobarbital.

ACKNOWLEDGMENTS

We aregratefultoDr. SusanLudmen,Dr. EllenShulman-Roskes,Dr.JohnHilton, andDr. David J. Waxman(BostonUniversity) for manyhelpful andscientific discussions, Dr. Thomas R. Koch from Maryland Medical Laboratory Inc. for his generous support in arranging the assay of plasma phenytoinandphenobarbitalconcentrations,Ann Marie Huelskampfor her patienceinproviding patient-relatedinformation,and RosemaryClark for her excellentsecretarialsupport.

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1995;55:810-816. Cancer Res   Tian-Ling Chen, Jose L. Passos-Coelho, Dennis A. Noe, et al.   TransplantationChemotherapy followed by Autologous Bone Marrowwith Metastatic Breast Cancer Receiving High-Dose Nonlinear Pharmacokinetics of Cyclophosphamide in Patients

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