0090-9556/98/2605-0418– 428$02.00/0
DRUG METABOLISM AND DISPOSITION
Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics
Vol. 26, No. 5
Printed in U.S.A.
URINARY EXCRETION OF CYCLOPHOSPHAMIDE IN HUMANS, DETERMINED BY
PHOSPHORUS-31 NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
CLAIRE JOQUEVIEL, ROBERT MARTINO, VERONIQUE GILARD, MYRIAM MALET-MARTINO, PIERRE CANAL,
AND ULF NIEMEYER
Biomedical NMR Group, Interactions Moleculaires et Reactivite Chinique et Photochinique Laboratory, Université Paul Sabatier (C.J., R.M.,
V.G., M.M.-M.), Centre Claudius Regaud (P.C.), and ASTA Medica AG (U.N.).
(Received September 30, 1997; accepted January 28, 1998)
This paper is available online at http://www.dmd.org
ABSTRACT:
amounts of phosphorylated metabolites excreted in 24-hr urine samples were much higher after the second CP dose (37%) than after the
first (20%), suggesting autoinduction of CP metabolism. CXCP and its
two degradation products (accounting for 7–10% of CXCP) were by
far the major metabolites (11.5 and 23% after the first and second
doses, respectively). DCCP plus its degradation products and alcophosphamide each represented 2–3% on the first day of treatment
and 5% on the second day of treatment. Levels of PM and its degradation products were extremely low (0.3 and 0.6% after the first and
second CP doses, respectively), as were those of ketophosphamide
(0.4 and 0.6% on the first and second days of treatment, respectively).
We noted only modest interpatient variation in excreted levels of CP
and all of its metabolites.
CP1 was introduced in tumor therapy in 1958 and is currently the
most widely used alkylating agent in human drug therapy (Brock,
1989; Sladek, 1994). As shown in fig. 1, CP is a prodrug that requires
biotransformation to become cytotoxic (Moore, 1991; Sladek, 1988,
1994). Activation as well as detoxication pathways are mediated by
hepatic CYP enzymes. Multiple CYP forms, including CYP3A4,
CYP2B6, CYP2C8, and CYP2C9, are capable of activating CP in
human hepatocytes (Chang et al., 1997), whereas CYP3A enzymes
catalyze .95% of the CP detoxication reaction in rat liver microsomes (Yu and Waxman, 1996) and CYP3A4 is the major enzyme
involved in CP detoxication in human liver microsomes (Bohnenstengel et al., 1996). First, hydroxylation of the oxazaphosphorine ring at
the carbon-4 position (activation pathway) leads to the formation of
OHCP, which exists in equilibrium with its ring-opened tautomer
AldoCP. Spontaneous b-elimination of urotoxic acrolein (Brock et al.,
1979) from AldoCP yields PM, the active alkylating species. OHCP
may be partially deactivated to KetoCP by an alcohol dehydrogenase.
AldoCP may be either oxidized to inactive CXCP by an aldehyde
dehydrogenase or reduced to AlcoCP by an aldehyde reductase
(Sladek, 1994) (fig. 1). Second, N-dechloroethylation of CP (detoxification pathway) produces DCCP and chloroacetaldehyde, a compound that may be responsible for the oxazaphosphorine-induced
neurotoxicity, urotoxicity, and cardiotoxicity (Goren et al., 1986; Pohl
et al., 1989; Joqueviel et al., 1997b).
Extensive pharmacokinetic data on CP itself have been published
and reviewed by Sladek (1988, 1994) and Moore (1991). However,
much less is known about the pharmacokinetics of CP metabolites. In
many of the early pharmacokinetic studies, total alkylating activity
was measured in the biofluid of interest by a colorimetric method
based on the ability of the alkylating species to react with NBP
(Friedman and Boger, 1961). This so-called NBP-alkylating metabolites assay (CP is devoid of alkylating activity) (Bagley et al., 1973)
does not provide information about specific metabolites, because all
of the alkylating agents present in the sample are measured indiscrim-
This study was supported by grants from the Association pour la Recherche
sur le Cancer (Grant 6635) and Ligue Nationale Française contre le Cancer
(Comité des Hautes-Pyrénées). This study was presented in part at the 88th
Annual Meeting of the American Association for Cancer Research (San Diego, CA,
April 12–16, 1997).
1
Abbreviations used are: CP, cyclophosphamide; CYP, cytochrome P450;
OHCP, 4-hydroxycyclophosphamide; AldoCP, aldophosphamide; PM, phosphoramide mustard; KetoCP, ketophosphamide; CXCP, carboxycyclophosphamide;
AlcoCP, alcophosphamide; DCCP, dechloroethylcyclophosphamide; NBP, 4-(pnitrobenzyl)pyridine; NNM, nor-nitrogen mustard; DDCCP, didechloroethylcyclophosphamide; IF, ifosfamide; Cr(acac)3, chromium(III) acetylacetonate; MPA,
methylphosphonic acid; 9-OdAP, oxadiazaphosphacyclononane; PAE1, phosphoric acid ester 1; PAE2, phosphoric acid ester 2; PAmA, phosphoramidic acid;
DCPM, dechloroethylphosphoramide mustard; PAEAc, phosphoric acid ester of
3-hydroxypropanoic acid; PAEAm, phosphoric acid ester of 3-hydroxypropanamide; PAmAE1, phosphoramidic acid ester 1; 6-OAP, oxazaphosphacyclohexane; PAmAE2, phosphoramidic acid ester 2; RT, repetition time.
Send reprint requests to: Dr. R. Martino, Groupe de RMN Biomédicale,
Laboratoire des IMRCP, Université Paul Sabatier, 118, route de Narbonne, 31062
Toulouse, France.
418
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Phosphorus-31 NMR spectroscopy was used to analyze urine
samples from patients treated with cyclophosphamide (CP) on 2
consecutive days. CP and all of its known phosphorylated metabolites except the tautomeric pair 4-hydroxycyclophosphamide/aldophosphamide, i.e. carboxycyclophosphamide (CXCP), dechloroethylcyclophosphamide (DCCP), alcophosphamide, ketophosphamide,
and phosphoramide mustard (PM), were determined. Several other
signals corresponding to unknown CP-related compounds were observed. Seven of them were identified; all were hydrolysis products of
CP or its metabolites (one from CP, two from CXCP, three from
DCCP, and one from PM). Twenty-four-hour urinary excretion of unmetabolized CP was not significantly different on the first (17% of the
daily administered dose) and second (16%) days of treatment. The
419
CYCLOPHOSPHAMIDE METABOLISM IN PATIENTS
31
P NMR.
Metabolites that are also degradation products, i.e. spontaneously formed, are shown in boxes. The names of the new metabolites found in this study are underlined.
Previously identified metabolites are named using their classical designations. The acronyms used for naming the new compounds are derived from the structural formulae,
with the number indicating the ring size (nine- or six-membered).
inately (Bagley et al., 1973; Fuks et al., 1981; Egorin et al., 1982;
Wilkinson et al., 1983).
Until recently, there was no single method available for the specific
determination of CP and its main metabolites in body fluids. The
TLC-photographic densitometry technique described by Hadidi and
Idle (1988) and modified by Boddy et al. (1992) and Tasso et al.
(1992) was the first method that reliably detected, in a single assay,
CP, CXCP, DCCP, and KetoCP, with concentration thresholds ranging from 2 to 8 mM, depending on the compound (Tasso et al., 1992).
However, contrary to claims made in the original report by Hadidi and
Idle (1988), PM and its cleavage compound NNM cannot be assayed
by this method (Yule et al., 1993), which involves solid-phase extraction of the sample and NBP derivatization. Almost all of the
studies on CP metabolism have used this method (Hadidi et al., 1988;
Boddy et al., 1992; Tasso et al., 1992; Yule et al., 1995).
Momerency et al. (1994) described a GC/MS method that determines accurately and with very high sensitivity ('2 nM) CP, CXCP,
DCCP, KetoCP, AlcoCP, NNM, and its adduct with bicarbonate ion
in plasma, i.e. 3-(2-chloroethyl)-1,3-oxazolidin-2-one, but not OHCP
or PM. This method requires two differential extraction treatments as
well as one or two derivatization procedures, depending on the compound. To our knowledge, no pharmacokinetic studies have been
carried out using this method.
Chan et al. (1994) published a pharmacokinetic study of CP and its
metabolites using a GC/MS system and a stable isotope-labeled
method, which adequately quantified CP, AlcoCP, PM, OHCP/AldoCP tautomers, and NNM, with detection limits ranging from 0.07 to
0.2 mM, depending on the compound. This analytical method has the
advantage of compensating for any procedural loss or decomposition
during work-up, by addition of appropriate deuterium-labeled internal
standards. The drawback is that the deuterium-labeled derivatives
must be synthesized first. Moreover, this technique requires two
separate clean-up procedures for the samples and a derivatization
procedure.
Compared with these chromatographic methods, 31P NMR presents
significant advantages. It enables direct analysis of crude urine samples, avoiding the problems encountered during clean-up and derivatization procedures, as well as those stemming from the pH sensitivity
of many metabolites. It allows detection and quantification, in a single
assay, of all of the phosphorus-containing compounds, because only
the presence of a phosphorus atom is required for detection. The only
major difficulty is its relative insensitivity. Nevertheless, it has been
used for quantitative study of the metabolism of IF (Martino et al.,
1992; Gilard et al., 1993a). We report here a direct qualitative and
quantitative 31P NMR analysis of CP and its phosphorylated metabolites in the urine of four patients treated with CP, with elucidation of
the structures of seven unknown compounds.
Materials and Methods
Materials. CXCP, DCCP, AlcoCP, KetoCP, PM, and DDCCP were generously supplied by ASTA Medica AG (Frankfurt, Germany). CP and IF were
obtained from ASTA Medica (Bordeaux, France). Cr(acac)3 and MPA were
purchased from Spectrométrie Spin et Techniques (Paris, France) and Aldrich
(St. Quentin Fallavier, France), respectively. 9-OdAP, PAE1, and PAE2 (fig.
2) were synthesized as described by Gilard et al. (1994). PAmA, degradation
products of CXCP (PAEAc and PAEAm) (fig. 1), and DCPM
[P(O)(OH)(NH2)(NHCH2CH2Cl)] were obtained using the methods described
by Sheridan et al. (1971), Joqueviel et al. (1997a), and Wang and Chan (1995),
respectively.
PAmAE1 (fig. 2) was prepared by dissolving 15 mg of DCCP in 2 ml of
aqueous 0.1 M HCl. After a few minutes, the solution was neutralized with 1
M KOH. 31P NMR showed that DCCP was completely hydrolyzed into
PAmAE1 and PAE2. PAmAE1 was purified by HPLC under the following
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FIG. 1. Metabolism of CP, incorporating the new phosphorylated compounds found in urine using
420
JOQUEVIEL ET AL.
conditions: column, Lichrosorb-RP Select B (5 mm, 150 3 4 mm); UV
detection, 200 nm; eluant, water/acetonitrile, 98:2; flow rate, 0.7 ml/min. The
retention time of PAmAE1 was 5.6 min, whereas that of PAE2 was 3.2 min.
The fractions containing PAmAE1 were immediately frozen in liquid nitrogen,
freeze-dried, and stored at 280°C.
Although 6-OAP (fig. 2) was not isolated, it represents 75% of the reaction
mixture (as estimated by 31P NMR) after an aqueous solution of PAmAE1 is
maintained at pH 8 for 3 days at room temperature. PAmAE2 (fig. 2) was
prepared by dissolving 12 mg of DDCCP in 2 ml of water and adjusting the pH
to 4.9. After 1.5 hr at room temperature, the solution was neutralized with 0.1
M NaOH. 31P NMR showed that PAmAE2 represented '90% of the reaction
mixture. The structures of PAmAE1, 6-OAP, and PAmAE2 were characterized
by MS and 13C NMR.
Patients and Urine Sampling. Four women with breast cancer participated
in the study. The median age was 48 years (range, 37–57 years). The patients
received CP iv at a dose of 60 mg/kg/day (as a 3-hr infusion in 1 liter of
isotonic glucose) for 2 consecutive days. The uroprotector mesna (2-mercaptoethane sulfonic acid) was administered at a dose of 60 mg/kg/day over 5 hr;
its infusion was started 2 hr before the start of the CP infusion. Hydration of
patients (3 liters of isotonic glucose/m2/day) was started 2 hr before the
infusion of CP and was continued for the 2 days of CP treatment. The complete
treatment required administration of mitoxantrone at a dose of 45 mg/m2 on
day 1, CP on days 2 and 3, and melphalan (L-phenylalanine mustard) at a dose
of 140 mg/m2 on day 4. No other phosphorylated drug was administered during
the study.
Urine was collected, in 6-hr time periods, for 24 hr after the start of the two
CP infusions. Urine volumes were recorded, and then the samples were
immediately stored at 220°C until the end of the 24-hr collection period and,
after that, at 280°C until NMR analysis, which was performed within 1 month
for samples at pH ,6.0 and within 1.5 month for the other samples. Because
the patients exhibited marked diuresis (2.5–7.9 liters/day, primarily .5 liters/
day), urine samples were concentrated. Twenty milliliters of urine were freezedried and resuspended in an accurately measured volume of H2O ('6 ml), with
care being taken to wash any residual pellet several times. The pH of the urine
samples ranged from 5.1 to 7.9 and was little affected by the concentration
procedure.
31
P NMR Analysis. 1H-decoupled NMR spectra were recorded at 121.5
MHz with a Bruker WB-AM300 spectrometer, without nuclear Overhauser
effect. The magnetic field was shimmed on the free induction decay from H2O
in the sample. 31P NMR chemical shifts are reported in ppm with respect to
85% H3PO4, which was used as an external reference. Spectra were acquired
using 10-mm-diameter NMR tubes under the following instrumental conditions: probe temperature, 4°C (25°C during hydrolysis of CXCP and PM);
sweep width, 15,151 Hz; data points, 32,768 zero-filled to 65,536; pulse width,
5 msec (i.e. flip angle, '35°); RT, 3.08 sec or 6.08 sec for qualitative or
quantitative purposes, respectively; free induction decay, processed by exponential multiplication with a line broadening of 3 Hz; number of transients,
5300 – 8300. The urine samples were doped at saturation (about 3 mM) with
the paramagnetic agent Cr(acac)3 to shorten the T1 relaxation times of the
phosphorylated compounds. The concentrations of all of the compounds detected were measured by comparing the areas of their 31P NMR signals with
that of MPA, the standard for quantification, which was placed in a sealed
coaxial insert; all signals were expanded on a 12-Hz/cm scale. The areas were
determined after the different signals had been cut out and weighed. The
external standard [MPA in deuterated water that had also been doped at
saturation with Cr(acac)3 to shorten its T1 relaxation time, with the deuterated
solvent providing the field frequency lock for the spectrometer] was calibrated
against IF solutions of known concentrations, with recording conditions [pulse
width, 5 msec (i.e. flip angle, '35°); RT, 6.08 sec] set to produce fully relaxed
spectra (Martino et al., 1992).
This 31P NMR method was validated for quantification of IF and its
phosphorylated metabolites in biological fluids (Martino et al., 1992; Gilard et
al., 1993a). Because the immediate environment of the phosphorus atom in CP
and its phosphorylated metabolites is identical to that in IF and its metabolites,
the T1 values for CP and IF and derivatives differed little. We therefore
assumed that our previous NMR recording conditions (flip angle, '35°; RT,
6.08 sec) would yield fully relaxed spectra in which the peak areas were
directly proportional to concentration. This was verified for CP and CXCP,
representing cyclic and linear phosphorylated CP-related compounds, respectively. Recording the spectra of solutions of these two compounds under the
conditions described and with a longer RT (10.08 sec), with all other parameters left unchanged, did not alter the signal intensities.
The accuracy and precision of the 31P NMR assay were determined in
several experiments. With the recording time used in this study (9 –14 hr), the
accuracy and precision of seven assays of CP at 1023 M and of AlcoCP and
DCCP at 1024 M in human urine doped at saturation with Cr(acac)3 and
adjusted at pH 8 or 5 were less than 610%. Moreover, we prepared solutions
of CP and CXCP at known concentrations down to '1025 M and of DCCP at
a concentration of '5 3 1026 M in human urine doped at saturation with
Cr(acac)3, with the pH adjusted to 7.0. Three to five assays per sample and two
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FIG. 2. Hydrolysis pathway for CP at moderately acidic and slightly basic pH values and acid hydrolysis pathways for DCCP and DDCCP.
All compounds are represented in their neutral forms.
CYCLOPHOSPHAMIDE METABOLISM IN PATIENTS
Results
Identification of CP and Its Phosphorylated Metabolites in
Urine from Patients. 31P NMR spectra of '3-fold concentrated urine
samples from patients treated with CP at 60 mg/kg/day showed the
presence of up to 30 signals (fig. 3, A and B). All d values mentioned
below were measured at pH 5.8 and 4°C unless otherwise indicated.
Parallel analysis of urine samples ('3-fold concentrated) from
patients before CP infusion demonstrated the presence of the Pi signal
and two faint signals in the phosphomonoester region (2– 6 ppm) (fig.
3C). The d values for these peaks were pH dependent, i.e. 1.29 (Pi),
2.45, and 2.68 ppm at pH 5.8 and 3.38 (Pi), 4.78, and 5.17 ppm at pH
7.8. The signal at 2.68 ppm (or 4.78 ppm) was attributed to phosphorylethanolamine and that at 2.45 ppm (or 5.17 ppm) to glycerol-1phosphate, as determined by spiking urine samples, at both pH values,
with the standards. The possibility of interference of the NMR signals
of CP-related phosphorylated compounds with those of compounds
from endogenous metabolism arises only with Pi, by far the major
compound detected in urine. The two endogenous phosphomonoesters
were found only in low amounts and in only a few of the samples
analyzed.
To assign some of these signals, urine samples were spiked with CP
and its metabolites and degradation products of CP and its metabolites. The data are listed in table 1, and fig. 1 illustrates our results. CP
(16.24 ppm), CXCP (20.78 ppm), DCCP (15.73 ppm), and AlcoCP
(20.92 ppm) were detected in all of the samples. KetoCP (8.44 ppm)
was observed in most of the samples (24 of 27 with pH values of
#7.5). It was not detected in the five samples at pH 7.8 –7.9, probably
because it was not sufficiently stable at that pH. The cytotoxic
alkylating agent PM (12.94 ppm) was detected in urine from the four
patients but only in a few samples (7 of 32).
The signal at 9.17 ppm observed in all of the samples corresponds
to the nine-membered ring compound 9-OdAP, as demonstrated by
spiking with the standard. This compound results from hydrolysis of
CP. In this respect, we demonstrated recently that, from moderately
acidic to slightly basic pH values ('3.5– 8.5), hydrolysis of CP leads
to 9-OdAP, the phosphorus-nitrogen bond breakdown of which is acid
catalyzed, giving rise to PAE1 (fig. 2) (Gilard et al., 1994). Addition
of PAE1 to several urine samples produced a new signal resonating at
2.95 ppm at pH 5.4, demonstrating its absence in the urine samples.
In acidic medium, DCCP is hydrolyzed in a three-step pathway (fig.
2) (Gilard et al., 1993b). The first step is the breakdown of the
phosphorus-nitrogen bond of the oxazaphosphorine ring. In the second step, the linear phosphoramidic acid ester (PAmAE1) is cyclized
with concomitant breakdown of the phosphorus-nitrogen chloroethyl
bond, leading to the six-membered ring compound 6-OAP. The third
step is the breakdown of the phosphorus-nitrogen bond of 6-OAP,
yielding PAE2. Spiking urine samples with PAmAE1, 6-OAP, and
PAE2 demonstrated that the signal resonating at 9.49 ppm, which was
observed in most of the samples (24 of 32), corresponds to PAmAE1.
The signals at 6.43 ppm (pH 7.8) and 2.77 ppm correspond to 6-OAP
and PAE2, respectively; one or both signals were observed in all
samples except four, in which the presence of PAmAE1 was noted.
6-OAP and PAE2 can also be derived from acid hydrolysis of
DDCCP (formed from N-dealkylation of DCCP), according to the
same degradation pathway as for DCCP (fig. 2) (Gilard et al., 1993b).
The presence of DDCCP and its first degradation product, PAmAE2,
was examined in urine samples. Addition of these two compounds led
to the appearance in the 31P NMR spectrum of two new signals, at
17.62 ppm (DDCCP) and 10.72 ppm (PAmAE2), demonstrating their
absence in the samples. Therefore, the degradation products 6-OAP
and PAE2 were thought to be derived from hydrolysis of PAmAE1,
the first degradation product of DCCP.
The two signals at 1.64 ppm and 1.77 ppm at pH 5.5 and 25°C were
attributed to degradation products of CXCP because their intensities
increased with time, while that of the CXCP signal decreased. These
signals, which were observed in most of the samples (25 of 32), were
identified by spiking urine samples with the two isolated and characterized products derived from degradation of CXCP in acidic medium.
At 4°C and pH 5.8, the upfield signal (1.90 ppm) corresponds to
PAEAc and the signal at 2.07 ppm corresponds to PAEAm (fig. 1).
To characterize the 31P NMR signals produced by hydrolysis of
PM, patient urine samples at acidic and neutral pH were spiked with
authentic compound. After 13 hr at 25°C in the NMR probe, the signal
for PM was markedly reduced. Six signals at pH 5.5 and nine at pH
6.8, resulting from PM degradation, were observed, of which three
were present in the urine samples before spiking, i.e. 13.11, 3.11, and
21.87 ppm at pH 5.5 and 15.82, 14.85, and 21.31 ppm at pH 6.8.
Only the signal upfield from the H3PO4 external reference was identified by spiking with authentic material. It corresponds to PAmA (fig.
1), giving a signal at 22.63 ppm at pH 5.8 and 4°C.
PM and its degradation products were observed in 25 of 32 samples. PAmA was observed in 16 (of 18) of the samples with pH values
of #6.9. It was not detected in samples with pH values of $7.0. PM
degradation products resonating (at 4°C) at '13.2 ppm (and also, in
a few samples, at '3.4 ppm) for pH #6.3 or at '16.0 ppm and '15.1
ppm for pH $6.6 were observed in 18 samples, together with the
signals from PAmA in nine of the samples. These signals consistently
accompanied those of PM. Although these compounds were not
identified, those producing signals between 13 and 16 ppm, i.e. close
to the PM signal, were assumed to contain structures whose environ-
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or three samples per concentration were analyzed. The accuracy and precision
were less than 610% for concentrations of 5 3 1024, 1024, and 5 3 1025 M
and approximately 620% at 1025 M. At 5 3 1026 M, the signal-to-noise ratio
ranged between 2:1 and 4:1, and this concentration was the detection limit for
our spectrometer. The accuracy and precision were then approximately 630 –
35%. Only the signals for PAE2 (in one urine sample), PM or its degradation
products resonating at '13–16 ppm (in two urine samples), and unknown
compounds were detected at concentrations of ,1025 M. We therefore think
that the quantification of all compounds with concentrations of $5 3 1025 M
was accurate (less than 610%) and that for concentrations between 5 3 1025
and 1025 M was acceptable (approximately 6 20%). Only the quantitative data
for the few compounds whose concentrations were ,1025 M were estimates.
Because of the length of time required for quantitating CP and its metabolites (9 –14 hr), the NMR data were acquired in '2.5-hr blocks. These blocks
were then compared to check the stability of the phosphorylated compounds
detected during the period of NMR recording. There were no significant
differences with time even for the most acidic urine samples, in which
degradation was most rapid. Quantitation was therefore carried out using
spectra resulting from the sum of all blocks.
The 31P d values for the degradation products of CXCP (PAEAc and
PAEAm) are close to that of phosphate ion (Pi), and their signals were thus
observed as a foot on the strong signal for Pi. We verified that standard PAEAc
added (at a known concentration of '5 3 1025 M) to human urine with the pH
adjusted to 8 or 5 was quantified with accuracy and precision of less than
610% (mean of seven measurements at each pH).
We also verified that the 3-fold concentration of the urine samples did not
lead to any loss of CP and its metabolites. The amounts of CP, AlcoCP1CXCP
(whose signals were not separable in the crude urine sample because of the
high value for line broadening used to improve the signal-to-noise ratio of the
spectrum), DCCP, and 9-OdAP [the only phosphorylated compounds, apart
from Pi, detected in a crude 0 – 6-hr urine fraction (pH 5.2)] were within 610%
of those measured in the same sample after concentration. However, 11 other
CP-related phosphorylated compounds were detected in the concentrated sample, increasing by 41% the amount of CP metabolites detected in the fraction.
All analyses were therefore carried out with the concentrated urine samples.
421
422
JOQUEVIEL ET AL.
TABLE 1
31
P NMR chemical shifts of signals detected in urine samples from patients
treated with CP
Chemical Shiftsa
Assignmentb
Urine
Authentic Standard in Urine
ppm
20.92
20.78
16.24
15.73
13.24
12.94
9.49
9.17
8.44
6.43 (pH7.8)
3.36
AlcoCP
CXCP
DDCCP
CP
DCCP
Degradation product of PM
PM
DCPM
PAmAE2
PAmAE1
9-OdAP
KetoCP
6-OAP
Degradation product of PM
PAE1
PAE2
PAEAm
PAEAc
Endogenous Pi
PAmA
a
Chemical shifts are related to external 85% H3PO4 and are from urine samples at pH 5.8 and
4°C (unless otherwise indicated).
b
Signals were assigned by spiking urine samples (concentrated '3-fold) with authentic
standards.
ment around the phosphorus atom was not significantly different from
that of PM. They were thought to be the compounds formed by
successive hydrolysis of chloroethyl groups that had been identified
by Watson et al. (1985):
FIG. 3.
31
P NMR spectra of urine samples from patients treated with CP at a
dose of 60 mg/kg/day.
A, fraction collected 18 –24 hr after the start of the infusion on the first day and
concentrated 3.4-fold (pH 5.8). The signals at 13.24 ppm and 3.36 ppm are derived
from the degradation of PM but are still unidentified. The signals at 2.68 ppm and
2.43 ppm correspond to endogenous urinary compounds. B, fraction collected 0 – 6
hr after the start of the infusion on the second day and concentrated 3.6-fold (pH
7.8). The signal at 15.26 ppm is derived from the degradation of PM. C, control
urine sample concentrated 3.1-fold (pH 5.8). Chemical shifts (d) are related to
external 85% H3PO4.
The N-dechloroethyl analog of PM (DCPM) was not detected in urine,
inasmuch as its addition led to the appearance of a new signal at 12.51
ppm.
The samples in which one or more of the phosphorylated compounds derived from degradation of CXCP, DCCP, or PM were not
detected (15 of 32) had relatively large volumes (1700 –3700 ml),
because of the overhydrated state of the patients (10 of 15), or were
samples in which the concentrations of CP metabolites, especially
those of CXCP or DCCP, were low (5 of 15). This could explain why
the concentrations of these compounds lay below the detection threshold of the 31P NMR assay ('5 3 1026 M), despite the '3-fold
concentration.
31
P NMR Chemical Shifts of Phosphorylated Compounds as a
Function of Urine pH. It can be seen from the results presented in
table 2 that the d values for CP and most of its metabolites (CXCP,
DCCP, AlcoCP, 9-OdAP, PAmAE1, 6-OAP, and KetoCP) were altered little in the range of pH values found in the urine samples (pH
5.1–7.9) or at which the compounds were detected. The signals for
these compounds could thus be unambiguously assigned in all spectra.
On the other hand, the signals for Pi and all of the compounds of the
same type, i.e. the phosphoric acid esters PAE2, PAEAc, and
PAEAm, exhibited marked variability in d values in this pH range
('2.5–3 ppm), because of protonation of the phosphate group. It
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2.77
2.07
1.90
1.29
22.63
20.92
20.78
17.62
16.24
15.73
13.24
12.94
12.51
10.72
9.49
9.17
8.44
6.43 (pH7.8)
3.36
2.95 (pH5.4)
2.77
2.07
1.90
1.29
22.63
CYCLOPHOSPHAMIDE METABOLISM IN PATIENTS
TABLE 2
31
P NMR chemical shifts of compounds identified in the urine of patients, as a
function of urine pH
31
P NMR Chemical Shiftsa
Compound
pH 5.1–5.3b
pH 7.8–7.9b
ppm
AlcoCP
CXCP
CP
DCCP
PM
PAmAE1
9-OdAP
KetoCP
6-OAP
PAE2
PAEAc
PAEAm
Pi
PAmA
20.92 6 0.01 (5)c
20.76 6 0.01 (5)
16.24 6 0.10 (5)
15.72 6 0.01 (5)
12.94 (1) (pH 5.8)
9.46 6 0.01 (5)
9.11 6 0.02 (5)
8.42 6 0.01 (5)
6.30 6 0.03 (2) (pH 6.3–6.4)
2.01 6 0.11 (4)
1.56 6 0.05 (5)
1.58 6 0.03 (2)
1.01 6 0.03 (5)
22.69 6 0.06 (5)
20.92 6 0.01 (5)
20.80 6 0.05 (5)
16.24 6 0.12 (5)
15.73 6 0.01 (5)
14.17 6 0.02 (2)
9.48 6 0.04 (4)
9.17 6 0.05 (5)
8.72 6 0.03 (5) (pH 6.9–7.5)
6.37 6 0.05 (3)
4.30 6 0.04 (2) (pH 6.6–6.7)
4.57 6 0.07 (4)
4.52 6 0.10 (4)
3.33 6 0.08 (5)
21.86 6 0.01 (3) (pH 6.7–6.9)
should be remembered that the second pKa of phosphoric acid is 6.7.
Although the strong signal from Pi was readily identified, the signals
of the three other compounds were assigned only after addition of
standards to the urine samples at the different pH values.
The signals for PM and its degradation products in urine samples
observed at 4°C, apart from that for PAmA, were readily identified
from the data obtained by monitoring the degradation of PM at pH 5.5
and 6.8 at 25°C. Indeed, only PAmA exhibited a marked variation in
d values between 25°C and 4°C (Dd ' 20.7 to 20.9 ppm) in this pH
range. The signal was thus assigned only after addition of the standard
to several samples at 4°C. However, compared with phosphoric acid
ester compounds, the signal for PAmA was more readily assigned
because it appears in a region containing few other signals. Furthermore, there is a relatively small variation in d values ('0.8 ppm) in
the pH range where it is detected (5.1– 6.9), because of the fact that its
two pKa values (3.0 and 8.3 at 4°C) lie outside this range (Gamcsik et
al., 1993).
Influence of Urine pH on Levels of Hitherto Unidentified Degradation Products. Because all of the new compounds identified
were derived essentially from hydrolysis (in acidic medium) of CP
and some of its metabolites, we examined whether there was a
relationship between the levels of these compounds and the pH of the
urine samples. The percentage of 9-OdAP, with respect to CP, did not
depend on the pH of the urine. The marked differences (table 3) were
the result of the fact that, in the 18 –24-hr samples obtained on the
second day of treatment, the amounts of CP excreted were low and
highly variable, leading to marked variations in the levels of 9-OdAP,
i.e. 9.2% (pH 6.9) to 90.7% (pH 6.3). Apart from these four samples,
the levels of 9-OdAP did not depend significantly on the pH of the
samples ('8%) (table 3). The urinary excretion of 9-OdAP by the
patients represents 6.1% of the CP excreted on the first day of
treatment (5.2–7.6%, depending on the patient) and 7.1% excreted on
the second day (5.5–10.7%).
On the other hand, the percentages of the degradation products of
CXCP (PAEAc and PAEAm), with respect to CXCP, depended on the
pH of the sample, i.e. the lower the pH the higher the percentage,
which is in line with the known instability of CXCP in acidic medium
(Ludeman et al., 1992; Joqueviel et al., 1997a). Despite the precautions taken during collection and storage of the samples, the degradation products of CXCP represented '40% of CXCP at pH '5 and
'20% at pH '6. At pH values greater than pH '6, there was
relatively little degradation of CXCP in urine (table 3). Urinary
excretion of the degradation products of CXCP by the patients represented 10.4% (range, 1.6 –35.5%) of the CXCP excreted on the first
day of treatment and 6.6% (range, 3.2–13.6%) excreted on the second
day. The higher percentage observed on the first day was the result of
the fact that the samples collected in the first 12 hr were the most
acidic.
The percentages of the degradation products of DCCP, with respect
to DCCP, did not depend on the pH in the range of 5.8 –7.9. On the
other hand, the percentages were markedly higher at pH 5.1–5.3 (57%
of DCCP vs. '22% at pH $5.8) (table 3). The first intermediate in the
acidic degradation of DCCP, PAmAE1 (fig. 2), is hydrolyzed directly
to PAE2 at the most acidic pH values (5.1–5.8). In contrast, at pH
$7.5, PAmAE1 is hydrolyzed only to 6-OAP. At intermediate pH
values (5.9 – 6.9), we observed 6-OAP with PAE2 (in six of nine
samples) or PAE2 alone. Approximately 40% of PAmAE1 was hydrolyzed in the urine samples, i.e. 43.7 6 13.0% into PAE2 at pH
#5.8 (N 5 6), 37.5 6 5.6% into 6-OAP at pH $7.5 (N 5 5), and
17.8 6 12.8% into 6-OAP (N 5 6) and 18.5 6 9.2% into PAE2 (N 5
9) at pH 5.9 – 6.9. Inclusion of these DCCP degradation products led
to an 18.6% increase (range, 12.1–25.9%) in the amount of DCCP
excreted on the first day of treatment and only an 11.0% increase
(range, 7.5–22.1%) in that for the second day. This discrepancy stems
essentially from the fact that six of the eight urine samples in which
no degradation products of DCCP were detected were collected on the
second day of CP treatment.
Because PM was always observed with its degradation products in
the 13–16 ppm range, it was always quantified with them. PAmA is
the major hydrolysis product of PM at acidic pH. It was the only
hydrolysis product of PM detected in seven samples, whereas it
represented '57% of PM and its degradation products in nine other
samples. At pH $7.0, only PM and its degradation products with
signals at '15–16 ppm were detected. The ratio of PAmA to PM plus
PM degradation products at 13–16 ppm was thus a function of the
sample pH. For example, the ratio was 0.1 for one patient (the pH
values for most of the urine samples from this patient were $7.5) and
5.0 for another patient (the pH values for the urine samples obtained
on the first day of treatment ranged from 5.1 to 6.1).
Urinary Excretion of CP and Its Phosphorylated Metabolites.
Table 4 summarizes the data on the 24-hr urinary excretion of CP and
its phosphorylated metabolites after the start of each 3-hr CP infusion
for the four patients treated on 2 consecutive days. The measured
values for four successive 6-hr aliquots after the beginning of each CP
infusion were summed to give the total amounts of CP and metabolites excreted in each 24-hr period. Fig. 4 is a stacked bar graph
showing the urinary recovery of CP and its phosphorylated metabolites in each 24-hr period; it includes individual data and mean data.
The total urinary excretion of CP and its phosphorylated metabolites represented approximately 36% of the daily administered dose of
CP on the first day of treatment and 52% of the administered dose on
the second day (CP metabolites that could have been degraded into Pi
are not included in this value). This 43% increase resulted from the
83% rise in excretion of all phosphorylated metabolites of CP on the
second day of treatment.
A mean of '16% unchanged CP was excreted on day 1 and '15%
on day 2. The hydrolysis product of CP, 9-OdAP, represented '1% of
the delivered dose on both day 1 and day 2. This increased the
percentage of unmetabolized CP to approximately 17% of injected CP
on day 1 and approximately 16% on day 2. It can be seen, therefore,
that the amount of unmetabolized CP (either alone or together with its
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 18, 2017
a
Chemical shifts are related to external 85% H3PO4. Spectra were recorded at 4°C. Values
are mean 6 SD.
b
pH of urine samples analyzed, unless otherwise indicated.
c
Numbers in parentheses are the number of values.
423
424
JOQUEVIEL ET AL.
TABLE 3
Variations in the percentages of hydrolysis products of CP, CXCP, and DCCP as a function of the pH of urine samples
Amount
Compound
pH 5.1–5.3
pH 5.8–6.1
pH 6.3–6.7
pH 6.9–7.1
14.5 6 10.4 (5)a
9.4 6 2.0 (4)
40.7 6 28.8 (5)
57.0 6 38.4 (5)
6.4 6 2.7 (7)
6.4 6 2.7 (7)
20.9 6 12.7 (7)
20.0 6 15.4 (5)
21.3 6 28.8 (7)
7.3 6 2.4 (5)
8.2 6 5.7 (4)
27.5 6 23.1 (5)
9.1 6 3.1 (6)
9.0 6 3.4 (5)
5.0 6 1.7 (4)
21.3 6 7.9 (3)
pH 7.4–7.9
Overall Mean Unless Otherwise
Indicated
7.2 6 4.2 (7)
7.2 6 4.2 (7)
3.2 6 0.6 (5)
17.1 6 6.0 (6)
11.6 6 15.5 (32)
7.7 6 3.3 (28)
5.3 6 3.9 (13) (pH 6.3–7.9)
21.5 6 15.8 (19) (pH 5.8–7.9)
%
9-OdAP
9-OdAPb
PAEAc 1 PAEAm
Sum of DCCP degradation
products
Data are expressed as percentages (mean 6 SD) of the parent compound [CP for 9-OdAP, CXCP for the sum of CXCP degradation products (PAEAc 1 PAEAm), and DCCP for the sum of
DCCP degradation products] in each urine sample analyzed.
a
Numbers in parentheses are the number of samples.
b
Data for the 18 –24-hr urine samples collected on the second day of treatment are not included.
TABLE 4
Excretiona
Day 1
Day 2
%
Total
Unmetabolized CP
Total of metabolites
9-OdAP
CP 1 9-OdAP
CXCP
PAEAc 1 PAEAm
CXCP 1 PAEAc 1 PAEAm
DCCP
DCCP degradation products
DCCP 1 DCCP degradation
products
AlcoCP
KetoCP
PM 1 PM degradation
products
Othersb
Activation pathway
metabolitesc
Deactivation pathway
metabolitesd
36.4 6 8.2 (24.2–47.2)
16.0 6 3.3 (11.5–20.7)
20.4 6 4.9 (12.7–26.4)
1.0 6 0.2 (0.8–1.2)
17.0 6 3.4 (12.3–21.8)
10.4 6 3.6 (5.2–15.2)
1.1 6 0.6 (0.3–1.9)
11.5 6 3.0 (7.1–15.5)
2.9 6 0.9 (1.6–4.1)
0.5 6 0.2 (0.3–0.8)
3.4 6 1.1 (1.8–4.8)
52.0 6 8.3 (43.5–61.1)
14.7 6 2.5 (11.7–18.2)
37.3 6 6.2 (30.8–45.4)
1.1 6 0.1 (0.9–1.3)
15.8 6 2.4 (13.0–19.3)
21.9 6 4.2 (17.5–27.9)
1.4 6 0.6 (0.8–2.4)
23.3 6 3.7 (19.9–28.9)
4.5 6 1.7 (2.6–6.3)
0.5 6 0.1 (0.3–0.6)
5.0 6 1.7 (3.2–6.9)
2.3 6 0.7 (1.1–2.8)
0.4 6 0.2 (0.1–0.6)
0.3 6 0.1 (0.15–0.4)
4.6 6 0.7 (3.6–5.6)
0.6 6 0.2 (0.3–0.8)
0.6 6 0.2 (0.4–0.7)
1.6 6 0.3 (1.1–2.0)
14.5 6 3.5 (9.0–18.6)
2.3 6 0.7 (1.8–3.4)
29.0 6 4.4 (24.9–35.9)
4.3 6 1.0 (2.6–5.9)
6.0 6 1.7 (4.2–7.9)
Values are mean 6 SD of the percentage of the CP delivered dose. The ranges are shown
in parentheses.
b
Includes all unknown phosphorylated compounds detected that were not present in blank
urine samples.
c
Includes CXCP, AlcoCP, PM, KetoCP, and their degradation products.
d
Includes DCCP and its degradation products as well as 9-OdAP, the hydrolysis product of
CP.
a
degradation product 9-OdAP) did not differ significantly ( p . 0.1)
between the first and second days of treatment.
The major metabolite ('12% on day 1 and '23% on day 2) was
CXCP, taking into account its degradation products (PAEAc and
PAEAm). DCCP is the major metabolite in the deactivation pathway
of CP, accounting for 2.9% (3.4%, including its degradation products)
on the first day of treatment and 4.5% (5.0%, including its degradation
products) on the second day. AlcoCP was excreted at similar levels,
i.e. 2.3% on day 1 and 4.6% on day 2. These percentages are
significantly higher than those for KetoCP and PM, which accounted
for ,0.5% on day 1 and 0.6% on day 2. Excretion of the other
unknown phosphorylated compounds derived from CP amounted to
1.6% (day 1) and 2.3% (day 2) of the injected dose. The urinary
excretion of the metabolites of the activation pathway (CXCP, AlcoCP, PM, KetoCP, and their degradation products) was significantly
higher ('3.5-fold on day 1 and '5-fold on day 2) than that of the
FIG. 4. Histograms of the urinary recovery of CP and its metabolites for 24 hr
after the start of each infusion (data for each patient and mean data).
The data are expressed as percentages of the delivered dose of CP. The values for
CP, CXCP, DCCP, and PM include their respective degradation products.
metabolites of the deactivation pathway (DCCP and its degradation
products as well as 9-OdAP, the degradation compound of CP).
Time Profiles for Excretion. The time profiles for excretion of CP
and its identified metabolites, including their degradation products,
and the sum of unidentified phosphorylated compounds derived from
CP are shown in fig. 5 as percentages of the daily administered dose
in each urine fraction. The same metabolites were found in each 6-hr
fraction. On the first day of treatment, the excretion of CP reached a
maximum (in three of four patients) in the 6 –12-hr fractions, whereas
CP metabolites (except KetoCP, which was at maximal levels in the
6 –12-hr fraction) peaked in the 12–18-hr fraction. Excretion subsequently decreased (more for CP than for its metabolites) in the
18 –24-hr fraction. The amount of excreted CP fell below that of the
sum of its metabolites in the 12–18-hr fraction. On the second day of
treatment, we found a marked increase in the urinary excretion of CP,
as well as its metabolites, in the 0 – 6-hr fraction. Excretion of CP and
its metabolites peaked in the first 6 hr (except for one patient, whose
excretion peaked in the 12–18-hr fraction) and then declined gradually
(except for KetoCP, the excretion of which was constant in the first
three fractions). Very small amounts of CP (0.4%) and small amounts
of CP metabolites (2.8%) were measured in the last collection period
(18 –24 hr). We thus concluded that excretion of intact CP is negligible 24 hr after the start of the 3-hr infusion and that the metabolites
of CP are also nearly all excreted in this period.
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 18, 2017
Cumulative urinary excretion of CP and its phosphorated metabolites, as
measured over 24 hr after the beginning of each 3-hr iv infusion of CP
CYCLOPHOSPHAMIDE METABOLISM IN PATIENTS
FIG. 5. Proportions of CP and its phosphorylated metabolites detected in urine,
expressed as percentages of the administered dose in each 6-hr fraction (mean of
values obtained for four patients).
Discussion
All of the phosphorylated CP metabolites thus far described, except
for the tautomer pair OHCP/AldoCP, which we did not attempt to
determine, could be assayed by 31P NMR. For accurate quantification,
OHCP and AldoCP, which are highly unstable, must be trapped by the
addition of appropriate stabilizing reagents, preferably at the bedside
(Chan et al., 1994; Ludeman et al., 1995).
Seven new phosphorylated compounds were identified in the urine
samples (fig. 1). 9-OdAP is derived from the hydrolysis of CP, and its
levels in urine were found not to depend on pH (table 3). Because CP
is little hydrolyzed at room temperature between pH 3.4 and pH 8.6
(#3% after 4 days at 20°C) (Gilard et al., 1994), 9-OdAP may be
derived from CP degradation in vivo rather than degradation during
collection and storage of urine samples.
PAEAc and PAEAm are derived from the hydrolysis of CXCP
(Joqueviel et al., 1997a), in amounts inversely proportional to the pH
of the urine samples (mean, 40% of CXCP at pH '5, ,10% at pH
.6.0) (table 3). This degradation may occur in vivo and during
collection of urine samples but not during storage at 280°C for ,6
weeks, in which time CXCP has been shown to be little degraded
(Joqueviel et al., 1997a). Therefore, urinary measurement of CXCP
does not reflect systemic production if the urine pH is acidic (#6).
PAmAE1, 6-OAP, and PAE2 are derived from the acid hydrolysis
of DCCP. Overall, these products made up approximately 20% of the
DCCP detected, and this level was found not to depend on urine pH
in the range of '6 – 8 (table 3). Because DCCP is stable for at least 3
days at room temperature at pH 6.5 (Martino et al., 1992), degradation
was thought to have occurred in vivo. On the other hand, the percentage of DCCP degradation was markedly increased at pH '5, which
may have occurred during collection and possibly also during storage
of the urine samples.
The absence of DDCCP and its primary hydrolysis product
(PAmAE2) indicated the absence of a sequential N-dechloroethylation
route for CP, in contrast to that observed for IF in both humans
(Martino et al., 1992; Gilard et al., 1993a) and rats (Wang and Chan,
1995). We failed to detect DCPM, a compound produced from DCCP
by the same process of metabolic activation as that transforming CP
into PM. This process has been demonstrated by Wang and Chan
(1995), who detected DCPM in the urine of rats treated with IF.
PM is extensively degraded in urine, and its degradation products
were detected with very little of the parent compound. Only the
compound derived from cleavage of the phosphorus-nitrogen bond
leading to liberation of NNM, PAmA, was formally identified. Overall, the degradation products of CP or its metabolites that were newly
identified in the present study accounted for '3% of administered CP
and made up approximately 15% of the excreted metabolites of CP on
day 1 and 10% on day 2.
Table 5 summarizes the data obtained for the urinary excretion of
CP and its metabolites using various analytical techniques, including
NMR. In our study, unmetabolized CP was the major compound
recovered in urine on day 1 of the treatment (16% of administered
dose) and the second major compound on day 2 (14.7%). These
amounts are in accord with the values reported by other authors
(11–20%) (table 5; see also Sladek et al., 1980; Fasola et al., 1991;
Chen et al., 1995) but are much higher than those reported by Jarman
et al. (1979) and Boddy et al. (1992), who administered much lower
doses of CP. The amount of CP excreted on the second day of
treatment was slightly less than that excreted on day 1, in agreement
with the findings of Fasola et al. (1991). For 19 patients treated with
a dose of 60 mg/kg/day for 2 days, those authors observed urinary
excretion of CP of 15.6 6 6.3 and 12.0 6 8.4% on days 1 and 2,
respectively. However, the difference between the 2 days was not
statistically significant in our study ( p . 0.1) and was at the limit of
significance in that of Fasola et al. (1991) ( p 5 0.03). In both studies,
some patients were found to excrete more CP on the first day of
treatment and others on the second day.
The major metabolite (and on day 2 the major compound) was
CXCP. It can be seen from table 5 that the levels we observed were
the highest of those reported for adults and close to the values
observed for children (Tasso et al., 1992; Yule et al., 1995), in whom
CP is metabolized more rapidly (Sladek, 1988, 1994; Moore, 1991).
However, it should be noted that our results included the degradation
products of CXCP, which were not included in other reports. The
rigorous analytical procedure (storage of samples at 280°C, storage
for no more than 6 weeks, and 31P NMR carried out at 4°C) limited
degradation to 1.1% of the injected dose on day 1 and 1.4% on day 2
(table 4). Under less stringent conditions, there might have been more
degradation, with a consequently lower percentage of excreted CXCP.
We observed less interpatient variation in the amounts of CXCP
excreted than in the studies listed in table 5, except for that of Jarman
et al. (1979). Those authors studied catheterized patients, whose urine
samples were left for a maximum of 2 hr at room temperature before
being cooled to 4°C to the end of the collection period (i.e. 24 hr) and
then stored at 230°C. Such collection conditions, which minimize the
degradation of CXCP, could account for the relatively low interpatient
variability. The small amounts of CXCP (5.5%) detected by those
authors most likely resulted from the significant degradation of the
compound upon extraction with ethyl acetate at pH 2, a pH at which
it is highly unstable. The marked variability among the other studies
might be derived, at least in part, from variable degradation resulting
from the pH of the samples, the conditions of urine collection and
storage, and the duration of urine storage before sample analysis. The
fact that our four patients were being treated for the same type of
cancer with the same therapeutic regimen could account for the low
interpatient variability we noted in the urinary excretion of CXCP and
the other metabolites (table 4). CP, which is known to be relatively
stable in urine, showed much lower interpatient variation than that
observed by Chen et al. (1995) or that in most of the studies listed in
table 5 or reviewed by Sladek (1994).
The amounts of DCCP excreted were comparable to those found in
children by Yule et al. (1995) and much higher than those detected by
Boddy et al. (1992) in adults and by Tasso et al. (1992) in children.
The percentage of excretion of AlcoCP was considerably higher
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 18, 2017
The histograms for CP, CXCP, DCCP, and PM include the proportions of
9-OdAP, PAEAm plus PAEAc, PAmAE1 plus 6-OAP plus PAE2, and PM degradation products, respectively.
425
16 6 5
(8–23)
62 6 10
(41–82)n
11
(3–19)f
16, 45, 62
60 or 75 mg/kg
1-hr iv infusion
Not reported
6–80 mg/kg
,10-min iv
infusion
24 hr
Radioactivity and
NBP assayd
34.2
(32.6–37.3)
12.0
(10.4–14.5)j
13.9
1.9
(1.2–2.8)h
20.3
(16.3–25.1)
24 hr
GC/MS/SIDe
3
26
Jardine et al.
(1978)a
11.8
6.6
1.1 6 0.6
(0.6–1.7)
5.2 6 1.7
(3.9–7.6)
5.5 6 1.3
(4.3–7.2)
24 hr
TLC/MS/SID
54–68
1g
iv bolus
4
Jarman et al.
(1979)a
6.4
9.0 6 5.7
(1.3–18.8)
36.2 6 17.8
(6.5–64.1)
2.6 6 1.9
(0.3–6.5)
1.9 6 3.1
(0.01–13.6)
0.8 6 0.9
(0.1–4.0)
3.3 6 3.7
(0.05–10.9)h
0.4 6 0.5
(0.02–2.1)
15–73
100–1080 mg
po or short iv
infusion
24 hr
TLC/PD
17
Boddy et al.
(1992)b
0.8 6 0.6
(0–2.1)k
23.4
18.5 6 16.1
(2.7–52.9)h
1.0 6 0.9
(0.4–3.9)
12.7 6 9.3
(2.8–31.4)
3.1 6 3.7
(0.05–10.1)
24 hr
TLC/PD
37–72
0.6–1.8 g/m2
1-hr iv infusion
14
Hadidi et al.
(1988)
37.4
(12.5–59.7)
18.5
53.7l
42.9l
3.0 6 2.0
0.4 6 0.4
39.0 6 30.0
10.8 6 4.2
Not reported
1 g/m2
20-min iv
infusion
24 hr
GC/MS/SID
4, 15, 17c
0.6–1.5 g/m2
1-hr iv
infusion
24 hr
TLC/PD
18.9
(5.4–43.2)
11.5
(3.4–22.3)
1.3
(0.3–3.2)
5.5
(0.7–8.5)h
0.5i
6
Chan et al.
(1994)
3
Tasso et al.
(1992)a
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 18, 2017
37–57
60 mg/kg/day on 2 consecutive days
3-hr iv infusion
24 hr after the start of each infusion
P NMR
3–18
557–1500 mg/m2
1-hr iv infusion
24 hr
TLC/PD
32.3 6 9.7
(23.0–52.4)
14.0
0.9 6 0.8
(0–2.0)
18.3 6 12.7
(5.4–43.2)
9.7 6 6.4
(4.0–22.3)
3.4 6 1.8
(0.3–6.0)
4
6
Day 2
14.7 6 2.5
(11.7–18.2)
23.2 6 3.7
(19.9–28.9)g
5.0 6 1.7
(3.3–6.9)g
0.6 6 0.2
(0.4–0.7)
0.6 6 0.2
(0.3–0.8)
4.6 6 0.7
(3.6–5.6)
37.3 6 6.2
(30.8–45.4)m
52.0 6 8.3
(43.5–61.1)m
Day 1
16.0 6 3.3
(11.5–20.7)
11.5 6 3.0
(7.1–15.5)g
3.4 6 1.1
(1.8–4.8)g
0.3 6 0.1
(0.1–0.4)
0.4 6 0.2
(0.1–0.6)
2.3 6 0.7
(1.1–2.8)
20.4 6 4.9
(12.7–26.4)m
36.4 6 8.2
(24.2–47.2)m
31
Present Study
Yule et al.
(1995)b
Data are expressed as percentages (mean 6 SD) of the CP-administered dose. Ranges are shown in parentheses.
a
These values are not reported in the paper but were calculated by us from the given data.
b
The values given by the authors were median values; mean 6 SD values given here were determined from the data reported in the paper.
c
Urinary recovery was reported only for the three patients receiving CP for the first time in the course of treatment, with administration in a single dose, and for whom urine collection was complete.
d 14
C-labeled CP was determined in the chloroform phase after urine extraction. Total alkylating metabolites were measured by the NBP colorimetric assay in crude urine samples, because CP did not react with NBP.
e
SID, stable isotope dilution; PD, photographic densitometry.
f
Values reported by Sladek (1994).
g
Because CXCP and DCCP are not stable at acidic pH, the amounts of their degradation products were included in the values reported.
h
Yule et al. (1993) subsequently demonstrated that PM was not reliably detected either with methods requiring its derivatization using diazomethane or with the TLC/photographic densitometry method.
i
KetoCP was detected for only one patient.
j
Those authors attributed the high levels of NNM, in part, to decomposition of other metabolites, especially CXCP, during sample preparation and storage.
k
Boddy et al. (1992) subsequently demonstrated that the NNM extraction procedure used was not reproducible and so NNM was not reliably assayed.
l
OHCP was also analyzed. Its amount (0.5 6 0.5%) was included in the total metabolites excreted value, as well as in the total excretion value.
m
These values included the amounts of 9-OdAP, the CP degradation product, as well as all unidentified CP-related compounds listed as “Others” in table 4.
n
Total radiolabeled excretion in 48 hr.
Total
metabolites
Total excretion
NNM
AlcoCP
KetoCP
PM
DCCP
Excretion (%)
Unmetabolized
CP
CXCP
Urine sampling
Analytical method
Number of
patients
Age (years)
Dose
Administration
Bagley et al.
(1973)
TABLE 5
Comparison of the urinary excretion of CP and its metabolites as reported in the literature and in the present study
426
JOQUEVIEL ET AL.
427
CYCLOPHOSPHAMIDE METABOLISM IN PATIENTS
levels in urine (Sladek, 1988) and the degradation of which could
therefore not account for the magnitude of the difference. A more
likely possibility is PM, the final hydrolysis product of which is Pi.
Several studies have shown that the urinary elimination of CP and
its metabolites is almost complete 24 hr after the start of treatment
(Bagley et al., 1973; Jardine et al., 1978; Sladek et al., 1980). This
was supported by the present findings. Only '6% of the injected dose
on day 1 and '3% on day 2 were recovered in the urine samples
collected 18 –24 hr after the beginning of the CP infusion. The marked
increase in urinary excretion of metabolites on the second day of
treatment (37.3% vs. 20.4% on day 1) (table 4) can be accounted for
by induction of the metabolism of CP. Autoinduction of the metabolism of CP was first described by Bagley et al. (1973), who noted
more rapid metabolism of CP from the second day to the fifth day of
treatment for patients receiving daily doses of CP (ranging from 6 to
80 mg/kg/day) for 5 consecutive days. The plasma half-life of CP was
found to be shorter and the levels of alkylating metabolites in plasma
higher on the fifth day than on the first day of treatment. These
findings were subsequently confirmed by numerous authors (Sladek et
al., 1980; Graham et al., 1983; Fasola et al., 1991) from the decrease
in the plasma half-life of CP. Fasola et al. (1991) showed that the
decrease in half-life (from 8.7 hr on day 1 to 3.6 hr on day 2) was not
accompanied by a significant drop in the urinary excretion of CP. This
indicated that the increase in the urinary excretion of phosphorylated
metabolites of CP is accompanied by induction of the metabolism of
CP, even in the absence of alterations in the excretion of CP. Very
recently, studies by Waxman and co-workers showed that CP induced
one of the CYP enzymes (CYP3A4) involved in CP 4-hydroxylation,
thus demonstrating an underlying metabolic basis for this autoinduction phenomenon (Chang et al., 1997).
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preliminary experiments that approximately one third of the PM was
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