[CANCER RESEARCH 38, 408-415, February 1978]

Quantitation by Gas Chromatography-Chemical lonization Mass

Spectrometry of Cyclophosphamide, Phosphoramide Mustard,and Nornitrogen Mustard in the Plasma and Urine of PatientsReceiving Cyclophosphamide Therapy1

I. Jardine,2 C. Fenselau,3 M. Appier, M-N. Kan, R. B. Brundrett, and M. Colvin4

Department ol Pharmacology and Experimental Therapeutics ¡I,J., C. F., M. A., M-N. K.J and Oncology Center ¡P.8. B., M. C.I, The Johns HopkinsUniversity School ol Medicine, Baltimore, Maryland 21205

ABSTRACT

Unambiguous and sensitive methods based on gaschromatography-chemical ionization mass spectrometry

have been developed to quantitate Cyclophosphamideand two alkylating and cytotoxic metabolites, phosphor-amide mustard and nornitrogen mustard. The levels ofthese materials have been determined in the plasma andurine of five patients receiving Cyclophosphamide, 60 or75 mg/kg i.v. Peak plasma levels of phosphoramide mustard of 50 to 100 nmoles/ml were found at 3 hr afterCyclophosphamide administration. Variable levels of nornitrogen mustard were found in the plasma. This productmay be arising in part from the decomposition of othermetabolites during sample storage and preparation.

INTRODUCTION

The metabolic activation of the widely used antineoplasticand immunosuppressive agent Cyclophosphamide is nowconsidered to occur as shown in Chart 1 (3, 4, 7, 8, 19).Carboxyphosphamide (VII) is the major urinary metabolitein animals and humans but does not possess significantcytotoxic activity (2, 19). 4-Ketocyclophosphamide (VI) isexcreted to a lesser extent than is Carboxyphosphamideand is noncytotoxic (2, 19). Much recent evidence hasstrongly implicated the route through 4-hydroxycyclophos-phamide (Ila) and aldophosphamide (lib) to phosphoramide mustard (///) as the important activating pathway of thedrug (7,8). Phosphoramide mustard was first demonstratedto be produced in mouse liver microsomal incubations ofCyclophosphamide (7) and subsequently was shown to bepresent in the plasma and urine of patients receiving Cyclophosphamide therapy (10). The alkylating properties ofintact phosphoramide mustard at physiological pH havebeen demonstrated (6), and it is probable that this compound represents the active alkylating moiety derived fromCyclophosphamide. However, the relative roles of phosphoramide mustard, 4-hydroxycyclophosphamide, and al-

1 This research was supported by USPHS NIH Grants CA-16783 and GM-

21248 and by grants from the Andrew Mellon Fund and the AmericanCancer Society, Maryland Division.

2 Present address: Department of Medicinal Chemistry and Pharmacog-

nosy. Purdue University School of Pharmacy and Pharmacal Sciences. WestLafayette. Ind. 47907.

1 Recipient of USPHS Research Career Development Award CA-00103.* Recipient of USPHS Research Career Development Award GM-70417.

Received May 27. 1977; accepted November 10. 1977.

dophosphamide in transport in the blood and into cellshave still to be clarified.

Several quantitative clinical studies have been conductedon patients receiving Cyclophosphamide (1, 3, 5, 15, 17).Generally, these analyses of blood and urine have beenmade for unchanged drug and total metabolites. In moststudies the parent drug has been measured by determiningthe organic solvent-extractable radioactivity. Metaboliteshave been assayed as nonextractable radioactivity or determined by the nitrobenzyl pyridine assay for alkylating activity (12). The results of studies with radioactive Cyclophosphamide are complicated by the fact that both ring- andside-chain-labeled cyclophosphamides have been used.When ring-labeled drugs are used, metabolites such asnornitrogen mustard and phosphoramide mustard are notmeasured.

In an attempt to understand more clearly the pharmacology of Cyclophosphamide and to approach detailed phar-macokinetic analysis of this important drug, it is essentialto be able to quantitate unambiguously Cyclophosphamideand all of its metabolites in the body fluids and tissues ofpatients receiving the drug. To this end we have developedmethodology to quantitate Cyclophosphamide, phosphor-amide mustard, and nornitrogen mustard in such samples(14). The methodology is directly extendable to the quanti-tation of other Cyclophosphamide metabolites.

We have applied these methods to measure the levels ofCyclophosphamide, phosphoramide mustard, and nornitrogen mustard in the plasma and urine of patients receivingCyclophosphamide therapy and have used the methodologyto examine the decomposition of Cyclophosphamide andsome of its metabolites to phosphoramide mustard andnornitrogen mustard and to examine some aspects ofprotein binding of these compounds.

MATERIALS AND METHODS

Materials. Cyclophosphamide monohydrate was generously provided by the Mead Johnson & Co., Evansville, Ind.Thin-layer chromatography of this material demonstratedthat it contained no phosphoramide mustard, nornitrogenmustard, or other aklylating impurities. Synthetic A/,/V-bis(2-chloroethyljphosphorodiamidic acid (phosphoramide mustard) as the cyclohexylamine salt, 4-ketocyclophosphamide,and Carboxyphosphamide were gifts from Dr. O. M. Friedman, Collaborative Research Corp., Waltham, Mass. Nornitrogen mustard was purchased from the Aldrich Chemical

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Quantitäten of Cyclophosphamide and Metabolites

Co., Milwaukee, Wis. d,-Nornitrogen mustard hydrochlo-ride was synthesized as previously described (14) by reduction of diethyliminodiacetate with LiAID4 to the corresponding diol with subsequent chlorination with thionyl chloride.d,-Cyclophosphamide monohydrate and d4-phosphoramidemustard (cyclohexylamine salt) were synthesized from d4-nornitrogen mustard by published methods (13). The purityof the deuterated standards was established by thin-layerchromatography and mass spectrometry.

Clinical Samples. Five patients were studied. A description of the patients and the doses of cyclophosphamidethat they received is shown in Table 1. For administrationto the patients, cyclophosphamide monohydrate was diluted with 100 ml of 0.9% sodium chloride solution andadministered within 1 hr of preparation.

All except Patient 1 received 20 or 40 mg of furosemidewithin 2 hr after the dose of cyclophosphamide. Patients 3and 4 were receiving allopurinol, 600 mg/day, prior to andduring the cyclophosphamide study, and Patient 4 wasalso receiving 45 mg of prednisone daily before and duringthe study. None of the patients received another antitumoragent during the study or during the preceding 2 weeks.With the exception of Patient 1, who had received a similardose of cyclophosphamide 7 days earlier, the patients werereceiving cyclophosphamide for the first time. All subjectshad normal blood urea nitrogen, creatinine clearance, serum alkaline phosphatase, and serum glutamic and pyru-vate oxaloacetic transaminase.

Yl

oldehydtC-0 ondo*« •¿�

CI .

CI '

•¿�za

Chart 1. Metabolism of cyclophosphamide.

Blood samples were collected in heparin, and the plasmawas separated immediately and frozen. Each patient voidedjust prior to the start of the cyclophosphamide infusion,and all urine was then collected on ice in 4-hr batches for24 hr. The volume and pH of these samples were recorded,and aliquots of each urine sample were frozen at the endof the 24-hr period.

Extraction and Derivatization. The plasma was thawedin lukewarm water, and to a 2- and a 1-ml sample wereadded aliquots of standard aqueous solutions of d,-phos-phoramide mustard and d,-cyclophosphamide plus d4-nor-nitrogen mustard, respectively. Similarly, standards wereadded to two 5- or 10-ml urine samples. The plasma andurine samples with added d4-nornitrogen mustard weretaken to pH 8.5 with dilute NaOH solution and extractedwith CHCI.',.The CHCI.-iwas dried with NaSO4, filtered, andevaporated. To the dried residue were added ethyl acetateand trifluoroacetic anhydride, and the samples were heatedto 70°for 20 min to effect derivatization. After blowing dry

in an N2 stream, the samples were reconstituted in ethylacetate, and 1- to 5-^.1 aliquots were injected in the gaschromatograph-mass spectrometer. The plasma and urinesamples with added d4-phosphoramide mustard were takento pH 4.5 with dilute HCI. They were then percolatedthrough prewashed (with methanol and water) columns ofAmberlite XAD-4 (Rohm and Haas, Philadelphia, Pa.), whichremoves the phosphoramide mustard from aqueous solution. The columns were washed with water, and the phosphoramide mustard was eluted with methanol. The methanol, which contains some water, was taken off by rotaryevaporation to complete dryness. Ethereal diazomethanewas then added, and the samples were allowed to stand for15 min with frequent stirring and scraping of the residueon the flask walls. The ether plus excess diazomethanewere then blown off, and the residue was redissolved inCHCI3 from which aliquots were injected into the gaschromatograph-mass spectrometer.

Estimates of the recovery of cyclophosphamide, nornitro-gen mustard, and phosphoramid mustard during the respective extraction procedures were made by carrying outthese procedures on known quantities of material. Therecoveries were greater than 90% for cyclophosphamideand nornitrogen mustard and approximatey 40% for phosphoramide mustard. While the trifluoroacetylation of cyclophosphamide and nornitrogen mustard appeared to go to

Table 1Patients and doses of cyclophosphamide administered

Patient Age (yr) Sex Diagnosis

Cyclophosphamide dose Time ofinfusion

mg/kg mg mmoles (hr)

123454762451662FMMFFCarcinomaoflungCarcinoma

oflungChronicmyelocyticleukemiaChronicmyelocyticleukemiaCarcinoma

oflung60607575604300370060003400250015.513.321.612.29.11.51.01.01.01.0

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completion, the methylation of phosphoramide mustarddid not. The use of deuterated compounds as internalstandards during the procedures compensates for lossesduring these steps.

For examination of the decomposition of cyclophospha-mide, 4-ketocyclophosphamide, and carboxyphosphamide

to phosphoramide mustard during the collection, extraction, and derivatization procedures described previously, aknown quantity of these materials with a standard amountof dj-phosphoramide mustard was added to separate non-

patient plasma and urine samples. The samples were frozen, stored overnight, thawed, extracted, and derivatized.Any phosphoramide mustard formed by decomposition ofthe added materials during these procedures could then bedetected and measured with the use of the d4 internalstandard.

Similarly, the decomposition of cyclophosphamide, 4-ketocyclophosphamide, carboxyphosphamide, and phosphoramide mustard to nornitrogen mustard during collection, extraction, and derivatization procedures was examined.

For examination of protein binding, a blood sample wasdrawn from Patient 5 2 hr after the start of a 1-hr i.v.

infusion of cyclophosphamide, 60 mg/kg. The heparinizedplasma was immediately frozen and stored. The plasmawas subsequently thawed, and a portion was stored at 4°.

The remaining plasma was filtered through a UM-10 ultrafil-tration membrane (Amicon Corp., Lexington, Mass.) at 4°.

The resulting ultrafiltrate and the plasma sample were thenanalyzed for cyclophosphamide, nornitrogen mustard, andphosphoramide mustard as described above.

Gas Chromatography-Mass Spectrometry. Analyses were

conducted on a Dupont 491 mass spectrometer coupledto a Varian 2700 gas Chromatograph via a glass jet molecular separator and equipped with a selected ion-recordingunit. The gas Chromatograph column was a 6-ft glass column packed with 3% OV-17 or 3% OV-101 on Supelcoportand operated at -130° for nornitrogen mustard-trifluoro-acetic acid, 220°for methylated phosphoramide mustard,and 260°for cyclophosphamide-trifluoroacetic acid. The

Chart 2. a, molecular ion regions of the isobutane chemical ionization mass spectra of standard and tetradeuteratedphosphoramide mustard as the trimethylated derivatives. Theprotonated molecular ions with their corresponding chlorineisotope species are at mie 263, 265, and 267. and at mie 267.269, and 271. respectively, o, 4-channel selected ion recordof the protonated molecular ion region of standard trimethylated phosphoramide mustard where the trace heights reflectthe chlorine isotope abundance. Even with the sensitivity onthe fourth channel for mie 269 at 50 times that of the other 3channels, no interference is apparent, c. 4-channel selectedion record of the protonated molecular ion region of thetetradeuterated material. The same ions are monitored as in6. It is observed that there is very slight overlap on the mie263 and 265 channels, even with both of these set at 50 timesthe sensitivity of the last 2 channels for mle 267 and 269. d.typical selected ion record of a derivatized patient plasmaextract to which deuterated phosphoramide mustard hadbeen added as internal standard. The concentration of phosphoramide mustard in the plasma sample is computed byusing the ratio of, e.g., mie 265:269. The gas Chromatographretention time and chlorine isotope ratios ensure that thecorrect peaks are being measured.

interface was heated to 280°,while the source temperaturewas maintained at 200°.Isobutane was the chemical ioniza

tion reagent gas.The gas Chromatographie profiles and the chemical ioni

zation mass spectra of cyclophosphamide-trifluoroacetic

acid, methylated phosphoramide mustard, and nornitrogenmustard-trifluoroacetic acid have been discussed previously (14). Chemical ionization produces essentially only aprotonated molecular ion species for each material withcorresponding chlorine isotopie ions at (M + 2)H+. For

example, the protonated molecular ion region for trimethylated phosphoramide mustard is shown in Chart 2a forboth the undeuterated and tetradeuterated species. Theselected ion record for these materials is shown in Chart 2,b and c. This shows that there is no interfacing overlap ation m/e 269 from the undeuterated species and negligibleoverlap (see "Calibration") at m/e 263 and 265 from the

deuterated species. These 4 ions are simultaneously monitored for patient plasma and urine samples to which deuterated internal standard has been added. A typical exampleis shown in Chart 2d. The gas Chromatograph retentiontime and chlorine isotope ratios ensure that we are measuring the correct ions. The ratio of appropriate unknown todeuterated sample ions (normally m/e 265:269) allowsquantitation of the unknown. Cyclophosphamide and nornitrogen mustard are assayed in similar fashion.

Calibration. It is essential in gas chromatograph-mass

spectrometer quantitative analyses to calibrate the experimental assays (9). Extensive calibration experiments wereconducted to determine the optimal and/or necessary ratioof internal standard to unknown to use, the limits tosensitivity that could be reached, and the precision andaccuracy of the assays for each of the 3 assayed materials.To this end standard mixtures of undeuterated to deuterated material were dissolved in plasma and urine samples,extraction and derivatization were carried out, and sampleswere injected on the gas chromatograph-mass spectrome

ter in at least triplicate.For these materials the best linearity and reproducibility

was obtained when the ratio of deuterated to undeuterated

0 02 ^_p£ 32 2 N_jjx3^CICHj—

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Quantitation of Cyclophosphamide and Metabolites

/. Jardine et al.

our decomposition studies indicated that at least some ofthe observed decomposition to nornitrogen mustard maybe occurring during the freezing and thawing procedure.In future studies this potential source of error will beeliminated by addition of the deuterated standard to theclinical sample immediately after collection. While the levels of phosphoramide mustard measured have not beenproduced artifactually, our decomposition experiments indicate that much of the nornitrogen mustard measuredmay be produced from decomposition of cyclophospha-mide and other metabolites, particularly carboxyphos-phamide, during the work-up. The fact that significantdecomposition of carboxyphosphamide to nornitrogenmustard occurs under mild conditions (freezing overnight,thawing, and brief exposure at pH 8.5) suggests to us thatsuch decomposition may be occurring in vivo.

Since we did not measure levels of 4-ketocyclophos-phamide or carboxyphosphamide, we could not measurethe contribution of the decomposition of these compoundsto our measurement of nornitrogen mustard. We haveelected to present the data that we have obtained and topoint out that the levels of nornitrogen mustard are probably artifactually high, especially in the case of the plasmameasurement. The measurements of nornitrogen mustardwere less consistent from patient to patient, perhaps dueto the decomposition shown above.

For the first patient study, plasma samples were measured at the end of a 1.5-hr infusion of cyclophosphamide,60 mg/kg, and at 1 and 8.5 hr after the end of infusion. Thevalues obtained from this study are shown in Table 3.

In further studies with 3 patients, more frequent bloodsamples were obtained, and urine levels of the 3 compounds were measured. The results of the plasma measurements from these patients are shown in Charts 4, 5, and 6.The first plasma sample from each of these 3 patients wastaken at the end of a 1-hr infusion of cyclophosphamide.Patient 2 received a dose of 60 mg/kg, and Patients 3 and4 received doses of 75 mg/kg. Patients 3 and 4 werestudied simultaneously, and the plasma and urine sampleswere handled identically.

In Patients 2 and 4, there was an initial rapid decline incyclophosphamide level during the first 5 hr, followed by aslower rate of decline. In Patient 3 the initial rate of declineof the cyclophosphamide level was less, and the 2 phaseswere less evident. The half-lives of cyclophosphamide inthe plasma after the cessation of the infusion were 4.4, 7,and 3.5 hr in Patients 2, 3, and 4, respectively. In all the

Table 3Plasma levels in Patient 1

Plasma levels

Cyclophospha- Phosphoramide Nornitrogenmide mustard mustard

Time" nmoles/ nmoles/ nmoles/(hr) ml mg/ml ml /¿g/ml ml ¿ig/ml

1.52.510269223817058215.910.45.41.32.31.230.16.38.55.31.11.5

eoo

500

4OO

3OO

Potient *2•¿�Cyclophosphomideo Nornitrogen Mustard«Phosphoramide Mustard

200-

Chart 4. Plasma levels (nmoles/ml) of cyclophosphamide, nornilrogenmustard, and phosphoramide mustard for Patient 2 determined from theend of a 1-hr cyclophosphamide infusion to 24 hr after the start of infusion.The time (hr) axis has been compressed between 8 and 24 hr, so the slopebetween these time points has no relevance. Dashed lines, no measurements.

Patient *3

•¿�Cyclophosphomideo Nornitrogen Mustard«Phosphoramide Mustard

" Time after start of 1.5-hr infusion of 4300 mg of cyclophospha

mide (60 mg/kg).

eoo r

500 •¿�

400-

300

200-

IOO-

Chart 5. Plasma levels (nmoles/ml) of cyclophosphamide. nornitrogenmustard, and phosphoramide mustard for Patient 3 determined from theend of a 1-hr cyclophosphamide infusion to 26 hr after the start of infusion.Dashed lines, no measurements.

patients cyclophosphamide was still present in the plasmaat 24 or 26 hr after the dose but was not detectable at 48and 69 hr.

The plasma levels of phosphoramide mustard showed aconsistent pattern in the 3 patients. The level of this metabolite rose to maximum at 2 to 3 hr after the start of infusion,remained elevated through 4 or 5 hr after the infusion, andthen slowly declined. The highest level seen was 100nmoles/ml, seen in Patient 4, the youngest of the patientsstudied. At 48 and 69 hr after drug administration, therewas no detectable phosphoramide mustard in the plasma.

In these 3 patients very high initial levels of nornitrogenmustard were measured, with a peak level of greater than500 nmoles/ml measured in Patient 2. As discussed abovethe nornitrogen mustard levels may be artifactually elevateddue to decomposition from other metabolites, particularlycarboxyphosphamide. However, the pattern of the plasma

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Quantitation of Cyclophosphamide and Metabolites

levels in these 3 patients is very consistent, with the peaklevel occurring at 2 hr after the start of the infusion and arapid decline in the plasma level occurring thereafter. (Apeak level was measured in Patient 3 at 2 hr, but the assaywas considered unreliable for technical reasons and is notincluded in the chart.) This consistent nornitrogen mustardplasma pattern indicates to us that either we are accuratelyfollowing the nornitrogen mustard plasma pattern or thenornitrogen mustard levels are closely reflecting the plasmalevels of another metabolite, such as carboxyphosphamide.

The results of the protein binding study done on theplasma from Patient 5 are shown in Table 4. Cyclophosphamide exhibited relatively little protein binding, but phos-

phoramide mustard was significantly protein bound, andnornitrogen mustard was extensively so. In control experiments it was shown that Cyclophosphamide, phosphoram-

ide mustard, and nornitrogen mustard in aqueous solutiondo not bind to the filter used. Because of the putativeinstability of the compounds being measured, these ultrafil-tration experiments were carried out at 4°and may not

accurately reflect the degree of protein binding at bodytemperature. Also, our results may have been altered bythe use of frozen plasma samples from the patient. In thispatient, as in Patient 1, the level of nornitrogen mustard at2 hr was much lower than that in Patients 2, 3, and 4.

A diagnostic problem necessitated the withdrawal ofcerebrospinal fluid from Patient 5 24 hr after the Cyclophosphamide infusion. A sample of this fluid was assayed

Table 524-hr urinary excretion of Cyclophosphamide and metabolites

Excretion (mmoles)

6OOPotient *4

•¿�Cyclophosphamideo Nornitrogen Mustard«Phosphoramide Mustard

Chart 6. Plasma levels (nmoles/ml) of Cyclophosphamide. nornitrogenmustard, and phosphoramide mustard for Patient 4 determined from theend of a 1-hr Cyclophosphamide infusion to 26 hr after the start of infusion.Dashed lines, no measurements.

Table 4Protein binding of Cyclophosphamide, phosphoramide mustard,

and nornitrogen mustard

Concentrations(nmoles/ml)Cyclophosphamide

Phosphoramide mustardNornitrogen mustardPlasma194

3343Ultrafil

trate"148

2014Protein

binding(%)24

3967

CyclophosphamidePhosphoramide mustardNornitrogenmustardTotalPatient

22.8(19.5)"

0.4 (2.8)1.4(10.4)4.6

(32.7)Patient

35.4(25.1)

0.3 (1.2)2.4(11.0)8.1

(37.3)Patient

42.0

(16.3)0.2 (1.8)1.8(14.5)4.0

(32.6)

Conditions of Ultrafiltration are described in the text.

" Numbers in parentheses, percentage of dose.

in the described way for Cyclophosphamide, phosphoramide mustard, and nornitrogen mustard. No Cyclophosphamide was detected in the cerebrospinal fluid at that time,but readily measurable quantities of phosphoramide mustard (1.8 nmoles/ml) and nornitrogen mustard (5.7 nmoles/ml) were present. Plasma levels at 24 hr were not measuredin Patient 5, but measurements at 24 or 26 hr in Patients 2,3, and 4 showed Cyclophosphamide levels of 0.6 to 11.6nmoles/ml, phosphoramide mustard levels of 6.6 to 11.9nmoles/ml, and nornitrogen mustard levels of 1 to 5nmoles/ml.

Urinary Excretion of Cyclophosphamide and Metabolites. The amounts of each of the 3 compounds measuredin the urine during the 24 hr following the dose of Cyclophosphamide are shown in Table 5. The percentage of theCyclophosphamide dose excreted as each of the compounds is similar in these 3 patients. The highest excretionof intact Cyclophosphamide (25.1%) was found in Patient3, who demonstrated persistently higher plasma levels ofthe parent compound than did the other 2 patients. However, the relatively high plasma levels of phosphoramidemustard seen in Patient 4 were not reflected in an increasedurinary excretion of this material. The sum of these 3compounds excreted in 24 hr accounted for between 32and 37% of the administered dose of Cyclophosphamide.Since very small quantities of these compounds were excreted during the last 4-hr collection period, it is unlikelythat a significant amount of the administered dose wasexcreted after the 24-hr collection period. The time course

of excretion of the materials measured in Patient 3 isshown in Chart 7. The excretions of Cyclophosphamideand phosphoramide mustard are initially high and decreasewith time, as might be expected from the plasma curves.However, the excretion of nornitrogen mustard is markedlyincreased at 8 to 16 hr after the dose, at a time after theplasma level has decreased markedly.

DISCUSSION

The initial levels of Cyclophosphamide in the plasmafound in these patients are in general agreement with theinitial levels of intact Cyclophosphamide found by Bagleyef al. (1) measuring chloroform-extractable radioactivityafter the administration of [14C]cyclophosphamide. When

the differences in doses of drug are considered, our resultsare also in agreement with the Cyclophosphamide levelsreported by Jarman ef al. (15), who measured Cyclophosphamide levels in plasma by isotope dilution of the probemass spectrum of plasma Cyclophosphamide.

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4 O

0-4 20-244-8 8-12 12-16 16-20

HOURS AFTER START OFCYCLOPHOSPHAMIDE INFUSION

Chart 7. Four- and 24-hr excretion totals in mmoles for cyclophosphamide. nornitrogen mustard, and phosphoramide mustard for Patient 3. •¿�.total urine volume in ml collected for each 4-hr period.

The plasma half-lives of the parent compound in Patients

2, 3, and 4 were measured at 3.4, 4.4, and 7 hr, respectively.This range of values is similar to those reported by Cohenef a/. (5), who found half-lives between 3 and 11 hr in 7

patients, and those values described by Bagley ef a/. (1),who found values ranging between 1.8 and 9.2 hr in theirstudy of 26 patients receiving cyclophosphamide. Similarfi,2 values were reported by Jarman ef a/. (15).

Patients 2, 3, and 4 excreted in the urine 19.5, 25.1, and16.3% of the dose of cyclophosphamide unchanged within24 hr of the administration of the drug. Again, these valuesare in agreement with those published by Cohen ef a/. (5),who found that 14.2% of the dose of cyclophosphamidewas excreted intact in the urine, and with the data ofBagley ef a/. (1), who found that an average of 10.1% of theparent drug was excreted in the urine during the first 24 hrwith negligible amounts being excreted after the first day.

The plasma levels of nornitrogen mustard and phosphor-

amide mustard are in agreement with the findings of Struckef a/. (20), who studied metabolites in the mouse afteradministration of cyclophosphamide and found high levelsof nornitrogen mustard and low but readily detectablelevels of phosphoramide mustard in the plasma. Moststudies of the metabolites of cyclophosphamide in humanshave measured either nonextractable radioactivity after theadministration of radiolabeled cyclophosphamide (17) orplasma alkylating activity (1, 3). In studies by Mouridsen efa/. (17), ring-14C-labeled cyclophosphamide was adminis

tered to patients, and the plasma levels of metaboliteswere determined as nonchloroform-extractable radioactiv

ity. This study of metabolite levels is not very relevant toour findings, since nornitrogen mustard and phosphoram

ide mustard would not be measured by this procedure.Brock et a/. (3) and Bagley ef a/. (1) have studied plasma

alkylating levels in humans after i.v. administration of various doses of cyclophosphamide. Bagley ef a/, found peakalkylating activities of 10 to 25 nmoles/ml occurring 2 to 4hr after doses of 60 mg/kg (1 patient) and 80 mg/kg (5patients). These values were obtained on plasma ultrafil-trates and were interpreted to represent the free plasmaalkylating metabolites. Bagley ef a/, also measured thealkylating activity of plasma extracts prepared by precipitation of the protein by perchloric acid. These values werehigher, and it was estimated on the basis of these data thatthe alkylating metabolites were 56% bound to the plasmaprotein. Brock ef a/, found peak plasma-alkylating levels of16 to 88 nmoles/ml in patients who received cyclophosphamide, 60 mg/kg i.v. These investigators studied perchloricacid-precipitated plasma and thus presumably were meas

uring total plasma alkylating metabolites. The time of thepeak alkylating activity varied from 1 to 4 hr after the dose,with most patients showing maximal alkylating activity at 2hr after the dose. We have measured alkylating activity inultrafiltrates of the plasma of patients after cyclophosphamide, 60 mg/kg, and have obtained peak values higherthan those obtained by Bagley ef a/, and similar to thevalues reported by Brock ef a/, for total plasma alkylatingmetabolites (M. Colvin, unpublished data). In both our studies and the reported data, there was marked variation inplasma alkylating levels between patients. Nornitrogen mustard, phosphoramide mustard, and carboxyphosphamideall give strong alkylating activity under the assay conditionsused in the above-mentioned studies. Therefore, the com

bined levels of phosphoramide mustard and nornitrogenmustard, plus the carboxyphosphamide that is presumablyin plasma but that we have not measured, should producehigher values of alkylating activity than those that havebeen observed. In an attempt to explain this discrepancy,we conducted studies in which quantities of nornitrogenmustard, phosphoramide mustard, and carboxyphosphamide approximately equal to the amounts of the phosphor-

amide mustard and nornitrogen mustard that we havemeasured in patient plasma were added to control plasma,and the alkylating activity of the plasma ultrafiltrate wasmeasured. When 500 nmoles of nornitrogen mustard, phosphoramide mustard, and carboxyphosphamide per ml wereadded to control plasma, the alkylating activity was 2, 6,and 0% of the alkylating activities obtained when the samequantities of the metabolites were added to 0.9% NaCIsolutions. The alkylating activities of these 0.9% NaCI solutions were not affected by ultrafMiration. From these experiments we conclude that measurements of plasma alkylatingactivity do not represent a direct measurement of either thesum of plasma alkylating metabolites or of phosphoramidemustard or nornitrogen mustard.

In the 1 patient whose plasma was studied, we found 24%of the cyclophosphamide to be protein bound. This value issomewhat higher than the 12 to 14% found by Bagley ef al.(1) for the protein binding of [14C]cyclophosphamide under

similar conditions. The values of protein binding of 39%for phosphoramide mustard and 67% for nornitrogen mustard that we found indicate that the polar metabolites aresignificantly bound to plasma proteins. This observation

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Quantitation of Cyclophosphamide and Metabolites

is in agreement with the finding by Bagley et al. (1) that 56%of the alkylating plasma metabolites were protein bound.Thus, these limited observations indicate that measurements of the binding of the specific metabolites to plasmaproteins will be important for complete pharmacokineticstudies of cyclophosphamide and its metabolites.

The significant and sustained levels of phosphoramidemustard observed in the patients studied here lend supportto the hypothesis that this material plays a role in thepharmacological activities of cyclophosphamide. Evidencehas been presented that this compound is the ultimatealkylating agent of cyclophosphamide (6). The question ofwhether phosphoramide mustard enters cells as such or istransported into cells in the form of a precursor, 4-hydrox-ycyclophosphamide or aldophosphamide, is not furtherclarified by these studies. Clearly, the plasma levels ofphosphoramide mustard reported here are cytostaticallysignificant, since Maddock ef a/. (16) reported that theconcentration of phosphoramide mustard corresponding to50% inhibition against a variety of mammalian cells inculture was from 30 to 80 nmoles/ml. The much lowercytotoxic dose that has been reported for activated cyclophosphamide (18) and 4-hydroxycyclophosphamide (21)suggests that 1 of the precursor compounds may be moreefficient at delivering phosphoramide mustard into the cell.However, the cytotoxic activity of phosphoramide mustardand 4-hydroxycyclophosphamide is similar in some systems(20).

The rates of conversion of 4-hydroxycyclophosphamideto aldophosphamide and of aldophosphamide to phosphor-amide mustard are not yet established. If these rates arevery rapid, then measurement of plasma phosphoramidemustard may be an effective monitor of the levels of theprecursor compounds in the plasma. On the other hand, ifeither 4-hydroxycyclophosphamide or aldophosphamide orboth have a significant existence time in aqueous solution,including plasma, then direct measurement of these compounds will be essential to the study of the pharmacoki-netics of cyclophosphamide and its metabolism in humans.Our recent detection of aldophosphamide in the plasma ofpatients treated with cyclophosphamide (11) indicates thatquantitation of this compound in vivo is possible.

The high levels of nornitrogen mustard seen in this studyare difficult to interpret. The decomposition in plasma andurine of cyclophosphamide, 4-ketocyclophosphamide, and,in particular, carboxyphosphamide to nornitrogen mustardsuggests that the high levels of this compound measuredmay be artifactual, produced during storage of the sampleand especially during the extraction procedure. However,the lability of carboxyphosphamide under the conditionsdiscussed above suggests to us that this decomposition ofcarboxyphosphamide to nornitrogen mustard in plasmaoccurs in vivo and that high levels of nornitrogen mustardare present in the body, especially in the urine. Since

nornitrogen mustard has a low therapeutic index (20) but istoxic, this metabolite may be contributing significantly tothe clinical toxicity of cyclophosphamide.

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2. Bakke, J. E., Feil, V. J., and Zaylskie, R. G. Characterization of theMajor Sheep Urinary Metabolites of Cyclophosphamide, a DefleecingChemical. J. Agr. Food Chem., 19: 788-790, 1971.

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8. Connors, T. A., Cox, P. J., Farmer, P. B., Foster, A. B., and Jarman, M.Some Studies of the Active Intermediates Formed in the MicrosomalMetabolism of Cyclophosphamide and Isophosphamide. Biochem. Pharmacol.,23: 115-129, 1974.

9. Falkner, F. C., Sweetman, B. J.. and Throck Watson, J. BiomedicaiApplications of Selected Ion Monitoring. Appi. Spectry. Rev., 10: 51-116,1975.

10. Fenselau, C.. Kan, M.-N. N., Billets, S . and Colvin, M. Identification ofPhosphorodiamidic Acid Mustard as a Human Metabolite of Cyclophosphamide. Cancer Res., 35: 1453-1457, 1975.

11. Fenselau, C., Kan, M-N. N., Subba Rao, S., Myles, A., Friedman, O. M.,and Colvin, M. Identification of Aldophosphamide as a Metabolite ofCyclophosphamide in Vitro and in Vivo in Humans. Cancer Res., 37:2538-2543, 1977.

12. Friedman, O. M., and Boger, E. Colorimetrie Estimation of NitrogenMustard in Aqueous Media. Anal. Chem., 33. 906-910, 1961.

13. Friedman, O. M., Boger, E., Grubliouskas, V., and Sommer, H. Synthesisof /V-Phosphorylated Derivatives of Nitrogen Mustards with Latent Cyto-toxicity. J.. Med. Chem.. 6: 50-58. 1963.

14. Jardine, I., Brundrett, R. B., Colvin, M., and Fenselau, C. Approachesto the Pharmacokinetics of Cyclophosphamide (NSC 26271): Quantitation of Metabolites. Cancer Treat. Rept., 60: 403-408, 1976.

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16. Maddock, S. L., Handler, A. H., Friedman, O. M.. Foley, G. E.. andFarber, S. Primary Evaluation of Alkylating Agent Cyclohexylamine Saltof N,N-bis(2-chloroethyl)phosphorodiamidic Acid (NSC-69945. OMF 59)in Experimental Antitumor Assay Systems. Cancer ChemotherapyRept.,50: 629-639, 1966.

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19. Struck, R. F., Kirk, M. C., Mellett, L. B.. El Dareer. S., and Hill, D. L.Urinary Metabolites of the Antitumor Agent Cyclophosphamide. Mol.Pharmacol., 7: 519-529, 1971.

20. Struck, R. F., Kirk, M. C., Witt, M. H., and Laster, W. R., Jr. Isolationand Mass Spectral Identification of Blood Metabolites of Cyclophosphamide: Evidence for Phosphoramide Mustard as the Biologically ActiveMetabolite. Biomed. Mass Spectrometry, 2: 46-52, 1975.

21. Takamizawa, A., Matsumoto, S., Iwata, T., Katagiri. K., Tochino, Y.,and Yamaguchi, K., Studies on Cyclophosphamide Metabolites andTheir Compounds. II. Preparation of an Active Species of Cyclophosphamide and Some Related Compounds. J. Am. Chem. Soc.. 95: 985-986.1973.

FEBRUARY 1978 415

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1978;38:408-415. Cancer Res I. Jardine, C. Fenselau, M. Appler, et al. Receiving Cyclophosphamide Therapyand Nornitrogen Mustard in the Plasma and Urine of PatientsSpectrometry of Cyclophosphamide, Phosphoramide Mustard, Quantitation by Gas Chromatography-Chemical Ionization Mass

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