Int Arch Occup Environ Health (2008) 81:285–293

DOI 10.1007/s00420-007-0211-2

ORIGINAL ARTICLE

Validation of urinary excretion of cyclophosphamide as a biomarker of exposure by studying its renal clearance at high and low plasma concentrations in cancer patients

Maria Hedmer · Peter Höglund · Eva Cavallin-Ståhl · Maria Albin · Bo A. G. Jönsson

Received: 10 April 2006 / Accepted: 18 May 2007 / Published online: 20 June 2007© Springer-Verlag 2007

AbstractObjectives Cyclophosphamide (CP) is an alkylating agentclassiWed as a human carcinogen. Health care workers han-dling this drug may be exposed during, e.g., preparation oradministration. Cyclophosphamide is readily absorbed byinhalation and by dermal uptake. A biomarker, CP in urine,has frequently been used to assess the occupational expo-sure to CP, but has not been fully validated. The aim of thisstudy was to investigate if the proportion of the CP dosethat is excreted in urine (renal clearance) is constant overdiVerent plasma drug concentrations and other pharmacoki-netic parameters, e.g., urine Xow.Methods Pharmacokinetics of CP were studied in 16breast cancer patients that were treated with postoperativeadjuvant chemotherapy including CP. Plasma and urinefrom the patients were collected at diVerent occasions up to12 days after the dose. Urine was collected during 4-h peri-ods and blood was sampled at the end of each period. Anal-ysis of CP was performed by liquid chromatographytandem mass spectrometry. The limit of detection for CP in

urine and plasma was 0.01 and 0.02 ng/ml, respectively.The precisions of the developed methods were determinedto ·8%.Results The administered doses of CP in absoluteamounts ranged between 800 and 2,240 mg. Mean renalclearance of CP was 8.6 (conWdence interval 6.5–10.7) ml/minand was not signiWcantly dependent of the plasma drugconcentration. However, a signiWcant correlation betweenrenal clearance and urine Xow was observed. There was alarge inter-individual variation in the plasma and urine con-centrations even when the same doses were given.Conclusions Cyclophosphamide in urine can be contin-ued to be used as a biomarker to monitor occupationalexposure to CP, however the inter-individual variability ofexcretion of CP in urine, and its dependency on urine Xowmust be taken into consideration in future applications.

Keywords Cyclophosphamide · Antineoplastic drug · Biological monitoring · Renal clearance · Urine Xow · Pharmacokinetics · Breast cancer patients · Risk assessment

Introduction

Cyclophosphamide (2-[bis(2-chloroethyl)amino]tetrahy-dro-2H-1,3,2-oxazaphosphorine 2-oxide; CP) is an alkylat-ing agent that is frequently used as an antineoplastic drug.It is administered as monotherapy or in combination withother drugs to treat neoplastic and non-neoplastic diseases.Cyclophosphamide is a prodrug and must be metabolizedthrough a group of cytochrome P450 (CYP) enzyme sys-tems in the liver, e.g., CYP2B6, CYP3A4, CYP2C9 andCYP2A6 before active alkylating metabolites can be gener-ated (Roy et al. 1999; Parkinson 2001). The metabolites aregenotoxic due to their ability to cross-link DNA and

M. Hedmer (&) · M. Albin · B. A. G. JönssonDivision of Occupational and Environmental Medicine, Department of Laboratory Medicine, Lund University Hospital, 221 85 Lund, Swedene-mail: maria.hedmer@med.lu.se

P. HöglundDivision of Clinical and Experimental Pharmacology, Department of Laboratory Medicine, Lund University Hospital, 221 85 Lund, Sweden

E. Cavallin-StåhlOncology, Division V, Department of Clinical Sciences, Lund University Hospital, 221 85 Lund, Sweden

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thereby cause DNA damages. CP is classiWed as carcino-genic to humans (IARC 1981, 1987).

Cyclophosphamide is widely used around the world.Health care workers handling this drug can be occupation-ally exposed and CP has been detected as a surface contam-inant in workplaces where antineoplastic drugs are used(Sessink et al. 1992a; McDevitt et al. 1993; Minoia et al.1998; Connor et al. 1999; Schmaus et al. 2002; Wick et al.2003; Hedmer et al. 2005). Amounts of CP have beendetected in urine (Hirst et al. 1984; Evelo et al. 1986; Ses-sink et al. 1992b, 1994; Ensslin et al. 1994; Minoia et al.1998; Turci et al. 2002; Pethran et al. 2003; Wick et al.2003), indicating that these workers are occupationallyexposed to CP. Absorption of CP occurs mainly throughskin or through inhalation of a gas phase or particulates.Since there is more than one exposure route for CP andextensive personal protection is used, it is suitable to use abiomarker for the determination of exposure. Biologicalmonitoring is also often easier to perform than ambientmonitoring. Thus, CP in urine has been used as a biomarkerof exposure, which allowed the internal dose of CP to beestimated (Hirst et al. 1984; Evelo et al.1986; Sessink et al.1992b; 1994; Ensslin et al. 1994; Minoia et al. 1998; Turciet al. 2002; Pethran et al. 2003; Wick et al. 2003).

The major fraction of CP in the body is eliminated byhepatic metabolism, but a small fraction is eliminated byrenal excretion of unchanged drug in urine. Approximately13% of the CP dose is excreted in urine (Sladek 1994), butindividual diVerences in the excretion of CP in urine in therange 3–36% have been reported (Bagley et al. 1973; Mil-stedt and Jarman 1982; Bailey et al. 1991; Ren et al. 1998;Joqueviel et al. 1998). The plasma half-life of CP isapproximately 5 h in humans during the Wrst days after thedose (Busse et al. 1997).

The current cancer risk assessments for health careworkers that are occupationally exposed to CP were madeby Sessink et al. (1995) and Sorsa and Anderson (1996). Inthose assessments renal clearance (CLR) of CP wasassumed to be independent of the plasma drug concentra-tion of CP. However, in pharmacokinetic studies performedby Busse et al. (1997, 1999) it was shown that the meanCLR was lower during treatment with conventional doses ofCP compared to high doses. This indicates that CLR of CPmight be dependent on the plasma drug concentration, i.e.that the proportion of the absorbed dose excreted in urinemay be lower for lower doses. If there is such dependence,biological monitoring of occupationally exposed workersusing CP in urine would underestimate their absorbeddoses, and thus, also the occupational risk at the low expo-sures.

The purpose of this study was to investigate CLR of CPat diVerent plasma drug concentrations of CP and therebyvalidate the use of CP in urine as a biomarker. This was

accomplished by studying patients treated with CP. In addi-tion, other parameters, e.g., the impact of urine Xow wasinvestigated.

Patients and methods

Patient population

A total of 16 women with breast cancer participated in thestudy. The patients were treated with postoperative adju-vant chemotherapy. Prior to entry into the study all patientsreceived both written and oral information about the study.The patients gave their written informed consent in accor-dance with the Helsinki Declaration. The study wasapproved by the Research Ethics Committee (LU 82–03) atLund University (Lund, Sweden).

Treatment

The adjuvant chemotherapy consisted of treatment everythird week. Dose calculations were based on body surfacearea. The chemotherapy protocol of conventional dose con-sisted of 600 mg/m2 of CP, 60 mg/m2 of epirubicin and600 mg/m2 of 5-Xuorouracil. Five of the patients receivedescalated doses of CP consisting of 1,200 mg/m2. The che-motherapy was given intravenously as an infusion througha permanent cannula. The conventional CP dose wasadministrated over a 30 min period while the escalated CPdose was administrated over 60 min. In addition, betameth-asone, mesna (2-mercaptoethanesulphonic acid) and trop-isetron hydrochloride were administrated to some of thepatients.

Blood and urine collection

Blood and urine samples were collected at three or fourdiVerent occasions up to 12 days after the chemotherapy.Blood samples were collected without using any perma-nent cannula due to the risk of contamination. Peripheral,venous blood was collected in 4 ml tubes (Vacuette®

K2EDTA, greiner bio-one, Kremsmüster, Austria). Theblood samples were centrifuged at 1,000g during 10 minto obtain plasma. The plasma was transferred to plastic testtubes with screw cap and stored at ¡20°C until analysis.At the Wrst sampling occasion an extra tube (3 ml Vacu-tainer LH PST™ II Plus, BD, Franklin Lakes, NJ, USA) ofblood was collected for analysis of plasma creatinine as ameasure of renal function. The liver function of thepatients was investigated by analysis of bilirubin,conjugated bilirubin, alkaline phosphatase, glutamyl trans-ferase, aspartate aminotransferase and alanine aminotrans-ferase in plasma.

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The patients collected urine during three or four 4-h peri-ods. Immediately before each period the patient voided andthat urine was discarded. All urine produced during each 4-h period was collected in 500 ml polyethylene bottles (Kau-tex Textron, Bonn, Germany). Patients voided at the end ofthe collection period and then a blood sample was drawn.The volume of the collected urine was measured and 20 mlaliquots of urine were stored at ¡20°C until analysis.

Analytical methods

Chemicals

Cyclophosphamide monohydrate (purity 99.5%) waspurchased from Sigma-Aldrich Chemie (Schnelldorf,Germany). 2H6-labelled cyclophosphamide (CP-D6; purity97%) was from Phychem (Bergisch Gladbach, Germany)and was used as internal standard. Ethyl acetate (HPLC99.8%) and methanol (HPLC) were from Lab-Scan (Dublin,Ireland). Acetic acid glacial (p.a. > 99.8%) and hydrochloricacid (p.a. 37%) were from Merck (Darmstadt, Germany).Trizma base (min. 99.9%) was from Sigma Chemicals (St.Louis, MO, USA). The water was puriWed in a Milli-RQWater PuriWer from Millipore (Billerica, MA, USA).

Standard preparation

Stock solutions of CP were prepared by dissolving weighedamounts in water. Several working solutions were madefrom these stock solutions by further dilution in water. Astock solution of CP-D6 was prepared by dissolvingweighed amounts in water, which were further diluted inwater. Standards up to 2,000 ng/ml were prepared. Plasmaand urine samples with higher drug concentrations werediluted.

Tris buVer preparation

A weighed amount of 12.1 g trizma base was dissolved inwater. The pH value of the solution was adjusted to eightwith 4 M hydrochloric acid. The solution was then dilutedto 100 ml with water.

Sample preparation

Aliquots of 0.5 ml of plasma were transferred into testtubes and 100 �l of internal standard (1.0 �g CP-D6/mlwater) as well as 100 �l of tris buVer were added. Five mil-liliter of ethyl acetate was added to the samples and theywere shaken with an IKA-VIBRA-VXR (IKA Labortech-nik, Staufen, Germany) for 5 min. Subsequently, they werecentrifuged at 1,500g during 5 min and the organic layerswere transferred to test tubes.

Aliquots of 1 ml urine were transferred to test tubes and100 �l of internal standard, 100 �l of tris buVer and 5 ml ofethyl acetate were added. The samples were shaken for5 min, centrifuged at 680g during 5 min and the extractswere transferred into new test tubes. The urine sampleswere extracted a second time with 5 ml of ethyl acetate.

Extracts from both plasma and urine samples were evap-orated to dryness under a stream of nitrogen gas at roomtemperature. The samples were dissolved in 150 �l 0.5%acetic acid, sonicated for 5 min and transferred into micro-vials. The samples were stored at 5°C until analysis. Allsamples were prepared in duplicate.

Standards of CP were prepared by adding 100 �l aliqu-ots of the working solutions to 0.5 ml drug-free humanplasma or 1 ml drug-free human urine, 100 �l internal stan-dard and 100 �l tris buVer, followed by the sample prepara-tion procedure.

Determination by liquid chromatography–mass spectrometry

The samples were analyzed by a Perkin-Elmer Series 200liquid chromatography (LC) system with a Series 200 auto-sampler (Applied Biosystems, Norfolk, CT, USA). The col-umn was a Genesis C18 (50 £ 2.1 mm) with a particle sizeof 4 �m (Jones Chromatography, Lakewood, CO, USA).The column outlet was coupled to an API 3000 triple quad-rupole mass spectrometer (Applied Biosystems/MDS-SCIEX, Toronto, Canada) equipped with an electrosprayionisation (ESI) source.

The LC conditions for analysis of CP were as follows:mobile phase A, 0.5% acetic acid; mobile phase B, metha-nol containing 0.5% acetic acid; time program, 0 min, 60%A/40% B; 6 min, 60% A/40% B; 8 min, 0% A/100% B;8.1 min, 60% A/40% B. During 2.9 min the column was re-conditioned (60% A/40% B). The Xow rate of the mobilephase was 0.2 ml/min and injection volume was 20 �l. Theion spray voltage was set to 3,000 V and the temperature to400°C. The instrument operated in positive ion mode usingmultiple reaction monitoring (MRM) at m/z 263.1/142.1 forCP and m/z 267.1/140.3 for CP-D6. The control fragmentm/z 261.0/140.3 was used for CP. Declustring potentials forCP and CP-D6 were 40 and 44 V, respectively. Collisionenergies were 31 V for CP and 34 V for CP-D6, respec-tively. Peak-area ratios between the analyte and the internalstandard were used for quantiWcation. The ratios betweenthe concentrations found by the analyte fragment and thecontrol fragment were not allowed to exceed 20%. If alarger deviation was observed the lowest value wasreported, which occurred very rarely.

To make standard curves, 1/x-weighted linear regressionwas used. Standard curves had correlation coeYcients>0.99 and showed good linearity. The limit of detection

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(LOD) was determined as three times the amplitude of thenoise.

Pharmacokinetic and statistical methods

From each patient three or four assessments of CLR weremade. The amount CP excreted during a sampling intervalwas calculated as the product of volume and concentrationof the urine. Plasma concentrations were analyzed at theend of each urine collection interval and the correspondingmid-point concentrations were calculated using the follow-ing assumed half-lives: 5 h up to 60 h from end of infusion;10 h in the interval 60–120 h from end of infusion; and 42 hafter 120 h from end of infusion. The CLR during eachinterval was then calculated as the amount excreted dividedby the product of calculated mid-point concentration andthe duration of the collection interval. The renal excretionrate of CP was calculated as the amount excreted through-out the duration of the collection period. Statistical analyseswere performed using SAS (version 8.2, SAS Institute,Cary, North Carolina, USA). Mixed model analyses withCLR as a dependent variable were carried out, using sub-jects as random eVects and diVerent pharmacokinetic vari-ables as covariates. The level of signiWcance was set atP < 0.05.

Results

The LOD for CP in plasma and urine was 0.02 and 0.01 ng/ml, respectively. The within-day precision of the developedmethods was evaluated by analysing ten plasma and urinesamples containing 1 ng of CP. The within-day precisionfor plasma and urine samples was determined to 4 and 2%,respectively. The between-day precisions of the methodswere studied by analysis of duplicates containing 0.5, 1, 5,50 and 100 ng/ml of CP. The duplicates were analyzed onthree diVerent days within a period of a couple of weeks.For both plasma and urine methods the between-day preci-sion were between 4–8%. Spiked plasma and urine samplescontaining 10 ng CP/ml were stored at room temperatureduring 24 and 48 h with no loss of CP. Spiked plasma andurine samples containing 10 ng CP/ml were also stored at¡20°C during a week and a month without any losses ofCP.

Patient characteristics, including renal and liver tests, arepresented in Table 1. The analyzed markers of hepatic andrenal functions were within the range of normal, or close tonormal. The administered doses of CP in absolute amountsranged from 800 to 1,200 mg (mean 1,000 mg) for patientsreceiving the protocol of conventional dose and from 2,000to 2,240 mg (mean 2,140 mg) for patients receiving theprotocol of escalated dose. An overview of the CP doses for

each patient is presented in Table 2. Also, their medicationswith other drugs during the study are shown.

The measured parameters from each of the patients canbe seen in Table 3. The Wnite time period of urine collectionranged between 175 and 330 (mean 240) min and urine vol-umes from 32 up to 840 ml were collected. Samplingoccurred from 20 to 285 h after ended treatment of CP. The

Table 1 Demographic mean characteristics of the participating pa-tients

Parameters Mean Range

Age (years) 52 38–61

Height (cm) 166 158–173

Weight (kg) 66 47–89

Body surface area (m2) 1.7 1.5–2.0

Plasma creatinine (�mol/l) 56 42–71

Plasma bilirubin (�mol/l) 12 6–24

Plasma bilirubin, conjugated (�mol/l) 2 1–5

Plasma alkaline phosphatase (�kat/l) 1.8 0.8–5.1

Plasma glutamyl transferase (�kat/l) 0.9 0.3–6.3

Plasma aspartate aminotransferase (�kat/l) 0.5 0.3–0.9

Plasma alanine aminotransferase (�kat/l) 0.7 0.3–1.7

Table 2 Doses of CP that the participating patients received and othermedications during the period of sample collection

a Escalated dose of CPb Used naturopathic drugs

Patient no.

CP dose (mg)

Other medications

1 1,200 Flunitrazepam, ranitidine

2 990 Prochlorperazine

3 1,010 Lorazepam, tropisetron

4 2,208a Lenograstim, levoXoxacin, prochlorperazine, tropisetron

5 2,090a Lenograstim, levoXoxacin, omeprazole, prednisolon, tropisetronb

6 2,000a Atenolol, levothyroxine

7 950 –

8 2,240a Furosemide, lenograstim, levoXoxacin, levothyroxine

9 980 Atorvastatin

10 950 Metoclopramide, tropisetron

11 800 CiproXoxacin, Xuconazole, lenograstim, nystatinb

12 2,170a –

13 1,050 Felodipine

14 920 –b

15 1,020 Paracetamol, prednisolone

16 1,150 –

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plasma concentrations of CP ranged between 0.02 ng/mland 6.1 �g/ml and the urine concentrations between0.29 ng/ml and 0.11 mg/ml. Plasma concentration at thesame sampling time varied ninefold between individualsreceiving the same doses of CP.

The calculated pharmacokinetic parameters for eachpatient are presented in Table 4. The calculated plasmadrug concentrations of CP in the middle of the Wnite timeperiod were between 0.02 ng/ml and 8.1 �g/ml. Area undercurve (AUC) and renal excretion rate of CP ranged between

Table 3 Measured parameters for each of the 16 participating patients

a Time after ended treatment with CP to the blood sampling

Patient no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1st sampling period (h)a 46 47 23 22 24 24 20 92 95 51 93 68 162 167 167 165

Plasma concentration CP (ng/ml) 61 45 1,200 3,500 760 2,200 6,100 5.6 1.2 320 4.6 7.5 0.12 0.10 0.17 0.19

Urine concentration CP (ng/ml) 540 980 22,000 93,000 4,700 17,000 110,000 81 16 2,300 69 92 1.0 1.1 1.8 3.8

Urine volume (ml) 240 67 130 130 360 360 100 470 110 400 180 190 64 330 32 150

2nd sampling period (h)a 95 94 119 92 95 96 92 140 143 99 141 170 186 211 189 189

Plasma concentration CP (ng/ml) 0.76 0.80 0.38 1.1 1.2 0.83 2.2 1.4 0.54 6.7 0.61 0.34 0.09 0.04 0.11 0.16

Urine concentration CP (ng/ml) 3.8 4.5 6.7 12 9.0 7.4 30 21 1.3 46 6.3 3.6 0.87 1.0 1.5 2.6

Urine volume (ml) 240 510 130 260 190 200 160 310 840 470 130 400 210 75 33 71

3rd sampling period (h)a 166 167 170 140 170 144 164 188 191 171 190 212 263 236 262 285

Plasma concentration CP (ng/ml) 0.20 0.20 0.19 0.68 0.97 0.53 0.51 0.93 0.33 0.54 0.31 0.24 0.03 0.02 0.06 0.03

Urine concentration CP (ng/ml) 0.66 0.93 3.4 7.0 3.0 2.1 1.9 9.7 1.4 1.8 2.5 1.6 0.29 0.65 0.62 0.44

Urine volume (ml) 450 490 140 100 170 230 160 310 390 210 300 400 410 180 45 400

4th sampling period (h)a 260 259

Plasma concentration CP (ng/ml) 0.40 0.21

Urine concentration CP (ng/ml) 2.5 1.3

Urine volume (ml) 430 450

Table 4 Calculated pharmacokinetic parameters for each of the 16 participating patients

a Time after ended treatment with CPb Concentration in the middle of the Wnite sampling period

Patient no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1st sampling period (h)a 46 47 23 22 24 24 20 92 95 51 93 68 162 167 167 165

Calculated plasma concentration (ng/ml) b

80 59 1,600 4,700 1,000 2,900 8,100 6.8 1.4 420 5.3 8.6 0.12 0.10 0.17 0.20

CLR (ml/min) 6.6 4.7 7.4 11 7.0 9.3 5.6 17 5.5 9.1 9.8 9.4 2.3 16 1.4 12

Urine Xow (ml/min) 0.98 0.28 0.55 0.55 1.5 1.6 0.40 1.4 0.47 1.7 0.75 0.88 0.27 1.4 0.13 0.64

2nd sampling period (h)a 95 94 119 92 95 96 92 140 143 99 141 170 186 211 189 189

Calculated plasma concentration (ng/ml)b

0.86 0.92 0.44 1.3 1.4 0.95 2.5 1.4 0.55 7.7 0.63 0.35 0.09 0.05 0.12 0.16

CLR (ml/min) 4.5 10 8.3 10 5.1 6.3 7.7 19 8.5 12 5.2 17 8.4 6.8 1.8 6.6

Urine Xow (ml/min) 1.0 2.1 0.54 1.1 0.77 0.81 0.65 1.3 3.5 2.0 0.53 1.7 0.89 0.31 0.14 0.41

3rd sampling period (h)a 166 167 170 140 170 144 164 188 191 171 190 212 263 236 262 285

Calculated plasma concentration (ng/ml)b

0.20 0.21 0.19 0.70 1.0 0.55 0.53 0.96 0.34 0.56 0.32 0.25 0.03 0.02 0.06 0.03

CLR (ml/min) 6.1 9.0 10 4.7 2.0 3.7 2.3 13 6.6 2.9 9.5 11 16 20 1.8 24

Urine Xow (ml/min) 1.9 2.0 0.58 0.47 0.66 0.94 0.64 1.3 1.6 0.88 1.2 1.7 1.7 0.75 0.19 2.0

4th sampling period (h)a 260 259

Calculated plasma concentration (ng/ml)b

0.41 0.22

CLR (ml/min) 11 11

Urine Xow (ml/min) 1.8 1.9

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5.7 min·ng/ml and 2.0 min·mg/ml and 0.12 ng/min and51 �g/min, respectively. The CLR and urine Xows werebetween 1.4 and 24 ml/min and 0.13 and 3.5 ml/min,respectively.

There was no signiWcant dependence between CLR of CPand plasma concentration of CP (P value 0.82), and theintercept (tmean CLR) was 8.6 (conWdence interval 6.5–

10.7; Fig. 1). When the plasma drug concentration valuesabove 10 ng/ml were excluded there was still no signiWcantdependence (P value 0.42) and the intercept was 8.3 instead.We have also used other cut-oV limits within our concentra-tion interval but still no signiWcance was obtained. Therewas, however, a signiWcant dependence between the CLR ofCP and the urine Xow (P < 0.01; Fig. 2).

Fig. 1 Plasma concentrations of CP versus CLR of CP for 16 patients.Vertical lines showing the plasma concentrations corresponding to theLOD of the analytical methods for plasma and urine are included. Alsoa vertical line indicating the plasma concentration corresponding to a

cancer risk of up to 400 cancer cases per million during a working pe-riod of 40 years is included. The mean CLR was used to calculate theplasma concentration from urinary levels

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Int Arch Occup Environ Health (2008) 81:285–293 291

Discussion

The major Wnding of this study was that we did not Wnd thatCLR was dependent on the plasma drug concentration ofCP. Since the proportion of the CP dose that was excretedin urine was constant, it is possible to continue to use thelevels of CP in urine as a biomarker of occupational expo-sure to CP. However, a dependence between urine Xow andCLR was discovered and there was a large inter-individualvariation in the plasma and urine concentrations even whenthe same doses were given. This should be consideredwhen using CP in urine as a biomarker of exposure.

The determination of CLR at low CP plasma levelsrequired analyses of extremely low biological concentra-tions of CP. Thus, highly sensitive analytical methods fordetermination of CP in plasma and urine were developedand validated regarding LOD, precision and stability. TheLODs in this study were lower compared to previous meth-ods for determination of CP in plasma (Sadagopan et al.2001; Liu et al. 2004) and urine (Sottani et al. 1998; San-nolo et al. 1999; Pethran et al. 2003; Kasel et al. 2004). Theprecisions of our methods were satisfactory and the stabil-ity of CP in the samples was high. With these methods it ispossible to perform biological monitoring of health careworkers involved in drug preparation and administration ofCP.

Since CP is classiWed as a carcinogen, it was not ethi-cally justiWable to study the pharmacokinetics on healthyvolunteers treated with low doses of CP. Instead, patientstreated with CP were monitored until the concentrations ofCP in urine and plasma were in the same low range as inoccupationally exposed workers. A number of diVerentcancer diseases can be treated with CP, e.g., leukemia,breast cancer, lung cancer, ovarian cancer, lymphoma, etc.Health care workers handling CP are mostly women rang-ing between 20 and 65 years of age. The design of the studywas chosen to mimic the real conditions and the investi-gated subjects were women 38–61 years old.

It has been reported that several drugs interact with theCYP enzyme systems in the liver and thereby alter thepharmacokinetics of CP by either induction or inhibition(Xie 2004). However, all investigated patients except onewere treated with drugs not known to alter these enzymesystems. Only one patient was treated with Xuconazole, adrug that might alter the pharmacokinetics of CP and sup-press the CYP genes for the enzyme CYP3A4 and CYP2C9(Marr et al. 2004; de Jong et al. 2005). Thus, the co-admin-istration of drugs probably did not aVect the results of thestudy.

Liver function may inXuence the metabolism of CP.Thus, if liver function is impaired, a smaller fraction of theCP dose is metabolized and a higher fraction of CP may beeliminated in the urine. The investigated patients were

treated with CP, epirubicin and 5-Xuorouracil one or sev-eral times before they participated in this study. These che-motherapy treatments may have an eVect on liver and renalfunction. From the result of the investigated markers ofliver and renal function it was observed that some of themarkers were slightly outside the normal range in a few ofthe patients. However, this small deviation was considerednot to have an impact on the result.

Blood samples were collected at the end of the samplingtime period. It was not practicable to collect blood in themiddle of the sampling period since the patients wereinvestigated in their homes. Therefore, the plasma drugconcentration in the middle of the sampling period was cal-culated.

In the literature it has also been reported that there is avariability in the CP pharmacokinetics, both inter-individ-ual and intra-individual variations of the CP pharmacoki-netics has been described (Moore et al. 1991; Sladek 1994;Chen et al. 1995; Busse et al. 1997; Batey et al. 2002). Weobserved both inter- and intra-individual variations of CLR

and urine Xow in this study. According to Xie (2004) thevariation in CP pharmacokinetics may be a result of bothenvironmental and genetic inXuences on the expression andactivity of CP-metabolizing CYP enzymes.

Previous pharmacokinetic studies of CP have reportedmean CLR’s during the Wrst 24 h after treatment to bebetween 15 and 20 ml/min in patients treated with conven-tional doses of CP (Chen et al. 1995; Busse et al. 1997; Renet al. 1998; Busse et al. 1999). During chemotherapy thepatients may have received intensive hydration, and thismight have eVected the CLR due to increased urine Xow andthereby an increased excretion of CP in urine (Chen et al.1995). In a study made by Busse et al. (1999) high urineXows, approximately 2–6 ml/min, were reported during24 h after the dose of CP for patients treated with conven-tional doses. In this study CLR seems to be lower, probablydue to the fact that the patient were studied between 20 and285 h after the CP dose and were not intensively hydrated.The urine Xow was in the range of 0.13–3.5 ml/min (mean1.1 ml/min) and values around 1 ml/min were considered tobe normal. Urine Xow can only have a substantial eVect onCLR if a drug is mostly reabsorbed (Rowland and Tozer1995). CLR of CP is thought to be predominantly the resultof glomerular Wltration and tubular reabsorption since CLR

of CP is much lower than the product of the fractionunbound and glomerular Wltration rate. However, it doesnot seem necessary to adjust for dilution in association withcollection of spot urine samples with, e.g., creatinine inurine, since this substance also co-variates with the urineXow.

The calculated elimination half-life of 5 h for CP up to60 h after treatment corresponds to the mean eliminationhalf-life reported by Busse et al. (1997). However, after the

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60 h, in this study, the elimination half-lives were consider-ably longer. Mean elimination half-lives were calculatedinstead of individual half-lives since we had only a fewobservations per subject.

A biomarker of exposure should ideally give a measureof the internal dose of the substance that has an eVect in thebody. In the case of CP it is the metabolite phosphamidemustard (PAM) that has antineoplastic activity. In a studyby Joqueviel et al. (1998) urine from patients treated withCP was collected during one and two days after treatment.One day after treatment CP was primarily excreted in urineas unmetabolized CP (mean 16%), followed by carboxy-phosphamide (CXCP; mean 10%) and dechloroethylcyclo-phosphamide (DCCP; mean 3%). Two days after the dosethe excretion of CXCP dominated the urine (mean 22%),followed by CP (mean 15%) and DCCP (mean 5%). Themean excretion of PAM and PAM degradation products inurine one and two days after the dose was 0.3 and 0.6%,respectively. It is therefore most suitable to use CP in urineto assess the short-term exposure to CP. Thulin et al. (1996)developed a method for analysis of hemoglobin adducts ofnornitrogen mustard, another metabolite of CP, but it isuncertain if this method is sensitive enough to measureoccupational exposure to CP.

In this study large individual variations in plasma andurine concentrations of CP can be seen although the samedoses of CP were administered. Thus, the biomarker CP inurine cannot estimate the internal dose of CP in exposedworkers with exactness since the doses either might beunderestimated or overestimated. However, the biomarkerreXects all exposure routes (dermal uptake, inhalation andingestion). The urine concentration makes it possible to cal-culate the plasma concentration and since the distributionvolume of CP is known (approximately 0.7 l/kg) theamount of CP in the body can be approximated (Sladek1994). Also, the use of CP in urine as a biomarker at grouplevel should be more accurate. From Fig. 1 it is evident thatCP in urine is a more sensitive biomarker than CP inplasma, and would therefore be preferable for the monitor-ing of occupational exposure.

The lowest concentration of CP that was measured inpatient urine in this study was 0.29 ng/ml. With a urineproduction of 1–2 l per day this urine concentration corre-sponds to an excreted CP amount of 0.29–0.58 �g CP perday. The cancer risk assessment for health care worker bySessink et al. (1995) was based on extrapolation from bothanimal and patient data and much more detailed than theassessment by Sorsa and Anderson (1996). Sessink et al.estimated that a mean daily excretion of 0.18 �g CP wouldgive up to 400 cancer cases per million during a workingperiod of 40 years. Thus, CLR in this study was investi-gated at urine and plasma concentrations similar to thoseof Sessink et al. A daily urinary excretion of 0.18 �g CP

corresponds to a plasma concentration of 0.015 ng/ml(Fig. 1).

An increase of up to 400 cancer cases per million inoccupationally exposed personnel may be regarded as a lowrisk of cancer, but it cannot be neglected. However, itshould be remembered that the risk assessment by Sessinket al. (1995) was based on exposure to CP only but in real-ity handling of antineoplastic drugs often involves severaldrugs with similar mechanisms of action, i.e. alkylatingproperties, and critical eVects (cancer, reproductive eVects).

In summary, CLR of CP was not dependent on theplasma drug concentration and it will therefore be possibleto continue to use CP in urine as a biomarker of occupa-tional exposure to CP. With the developed analytical meth-ods it will be possible to perform biological monitoring ofCP in occupationally exposed workers. However, both thelarge inter-individual variability of excretion of unchangedCP in urine and the Wnding that the excretion of CP isdependent on urine Xow has to be considered in futureapplications where CP in urine is used as a biomarker ofshort-term exposure to CP to estimate the absorbed dose ofCP.

Acknowledgments We would like to thank Ms Eva Assarsson, MsElse-Marie Åkerberg and Ms Inger Bensryd for their excellent medicalskills and Ms Gertrud Wohlfart for her excellent and skilful laboratoryassistance. Financial support for this study was obtained from theSwedish Council for Work Life and Social Research, the Swedish Re-search Council, the County Councils of Southern Sweden and the Med-ical Faculty at Lund University in Lund, Sweden.

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