[CANCER RESEARCH 48, 5167-5171, September 15. 1988]
Phenotypically Deficient Urinary Elimination of
Carboxyphosphamide afterCyclophosphamide Administration to Cancer
Patients1
Al-Hakam F. A. Hadidi,2 Carmel E. A. Coulter, and Jeffrey R.
Idle3
Department of Pharmacology and Toxicology, St. Mary's Hospital
Medical School [A. F. A. H., /. R. I], and Department of
Radiotherapy, St. Mary's Hospital
[C. E. A. C.I, London, United Kingdom
ABSTRACT
The 0-24-h urinary metabolic profile of cyclophosphamide was
investigated in a series of 14 patients with various malignancies
receivingcombination chemotherapy including i.v. cyclophosphamide.
This wasaccomplished using combined thin-layer
chromatography-photography-densitometry, which can quantitate
cyclophosphamide and its four principal urinary metabolites
(4-ketocyclophosphamide, nor-nitrogen mustard, Carboxyphosphamide,
and phosphoramide mustard). Recovery ofdrug-related metabolites was
36.5 ±17.8% (SD) dose, the most abundantmetabolites being
phosphoramide mustard (18.5 ±16.1% dose) andunchanged
cyclophosphamide (12.7 ±9.3% dose). The most variablemetabolite
was Carboxyphosphamide, with five patients excreting 0.3%dose or
less. These patients were termed low carboxylators (LC) andcould be
distinguished from high carboxylators (HC) by a carboxylationindex
(relative percentage as Carboxyphosphamide multiplied by 10).Mean
carboxylation indices for the LC and HC phenotypes were 3.4 ±2.6
and 151 ±115, respectively. There were no associations
betweenpatient age, sex, body weight, tumor type, or concomitant
drug therapyand carboxylation phenotype. Neither
4-ketocyclophosphamide nor nor-nitrogen mustard excretion differed
between LC and HC phenotypes;however, HC patients had a greater
excretion of cyclophosphamide (46.4±15.5 relative percentage) than
LC patients (19.4 ±12.6%). The DNAcross-linking cytotoxic
metabolite phosphoramide mustard was elevatedmore than 2-fold in
the LC (76.5 ±13.9%) compared with the HC (33.0±12.2%) phenotype.
It is concluded that these data represent the firstevidence of a
defect in cyclophosphamide metabolism, and it is proposedthat this
arises from a hitherto unrecognized aldehyde
dehydrogenasegenotype.
INTRODUCTION
Metabolic transformation is of paramount importance forthe
cytotoxic antineoplastic drug cyclophosphamide because itexerts
both its beneficial DNA-alkylating effect and its unwanted clinical
toxicity via metabolites. Variation between patients in the extent
of this metabolic activation would not onlybe of interest but would
also play a significant part in determining the outcome of therapy
with cyclophosphamide. Nevertheless, it is the qualitative pattern
of metabolism rather thandetailed quantitative studies which have
preoccupied investigators to date, primarii) as an attempt to
understand its mode ofaction and favorable therapeutic index.
As a consequence, the principal metabolic pathways undertaken by
cyclophosphamide are now well established (Fig. 1).Cyclophosphamide
is first converted to 4-hydroxycyclophos-phamide (1-3) by hepatic
cytochrome P-450 monooxygenases(4-6). Tautomerization of
4-hydroxycyclophosphamide yieldsthe ring-opened aldehyde metabolite
aldophosphamide (7-10)
Received 2/10/88; revised 6/15/88; accepted 6/21/88.The costs of
publication of this article were defrayed in part by the
payment
of page charges. This article must therefore be hereby marked
advertisement inaccordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
1Supported by grants from the Cancer Research Campaign (UK) and
The
Wellcome Trust.2Supported by The British Council and Yarmouk
University, Jordan. Present
address: Faculty of Medicine, Jordan University of Science and
Technology,Erbid, Jordan.
3Wellcome Trust Senior Lecturer. To whom requests for reprints
should be
addressed.
which acts as the branching point for either detoxication
toCarboxyphosphamide (2, 11, 12) or further activation to
thecytotoxic species phosphoramide mustard (13, 14), which
canitself be further cleaved to bis(2-chloroethyl)amine
(nor-nitrogen mustard) (7, 15, 16). Minor metabolites include
4-oxocy-clophosphamide (4-ketocyclophosphamide) (2, 12,14)
togetherwith the 4-hydroxycyclophosphamide dehydration product
im-inocyclophosphamide (not shown in Fig. 1) (17). Finally
andimportantly, the production of phosphoramide mustard
fromaldophosphamide yields an equimolar quantity of acrolein
(13,14, 18) thought to be responsible for the urotoxicity of
cyclophosphamide (19-22). Considerable evidence has not been
accumulated which implicates phosphoramide mustard ratherthan
4-hydroxycyclophosphamide, aldophosphamide, or nor-nitrogen mustard
as the ultimate alkylating and DNA cross-linking metabolite (23).
Although cyclophosphamide was originally synthesized (24) as a
prodrug for nor-nitrogen mustardrelease (25) within the tumor,
nor-nitrogen mustard is nowknown only to cause DNA-protein
cross-links (26).
Clearly, interindividual differences in the balance of
aldophosphamide metabolism to either phosphoramide
mustard(activation) or Carboxyphosphamide (detoxication) would be
ofconsiderable clinical importance. Until now, however, no
singleand simple method has been available for the
quantitativedetermination of cyclophosphamide metabolites in body
fluids.We have recently described (27) a combined TLC-PD"
method
which can determine cyclophosphamide, 4-Ketocyclophos-phamide,
Carboxyphosphamide, phosphoramide mustard, andnor-nitrogen mustard
in biological samples and have appliedthis in a reappraisal of the
quantitative nature of cyclophosphamide metabolism in cancer
patients. This paper reports thefinding of 5 cancer patients, from
a series of 14 who were giveni.v. cyclophosphamide, with a virtual
complete absence in urineof the "major" metabolite
Carboxyphosphamide with a conse
quent amplification of the activation pathway that yields
phosphoramide mustard.
MATERIALS AND METHODS
Authentic metabolites of cyclophosphamide were the gift of
Asta-Werke AC, Bielefeld, Federal Republic of Germany, and of
BoehringerIngelheim Limited, Bracknell, United Kingdom. Each
substance wasauthenticated by 'H-nuclear magnetic resonance and IR
spectroscopyat Asta-Werke AG and by elemental analysis in London,
details ofwhich are reported elsewhere (27). The TLC-PD method for
the determination of cyclophosphamide metabolites has been
described in detail(27). Briefly, urinary metabolites are extracted
onto a non-ionic polymeric adsorban), cltiled with methanol, and
separated with TLC. Visualization of drug-related spots is
accomplished by spraying the plateswith 4-(4-nitrobenzyl)pyridine,
an alkyl acceptor which gives a bluechromophore on alkalinization
(28). Because the blue spots deteriorateon standing, the TLC plate
is photographed within 10 s of spotdevelopment and the resultant
black and white print is scanned in adensitometer. The method is
sensitive down to 1 /ig/ml and linear up
4The abbreviations used are: TLC-PD, combined thin-layer
chromatography-photography-densitometry; LC, low carboxylator; HC,
high carboxylator; TLC,thin-layer chromatography; ALDH, aldehyde
dehydrogenase.
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DEFICIENT URINARY CARBOXYPHOSPHAMIDE EXCRETION
XCICH.jCH/
CICH,CH
OCH,CH;
OCH;CH
GKHfH
CP
P-450
4HCP
aCH,CH/PM +
CICHjCHA>NH
CICH,CH/COOH
NNMFig. 1. Metabolic transformations of cyclophosphamide «I')
Boldface ar
rows, activation pathways; open arrows, detoxication pathways.
For abbreviationssee Table 2. 4HCP, 4-hydroxycyclophosphamide; KP,
4-ketocyclophosphamide;AP, aldophosphamide; I'M, phosphoramide
mustard; ( 'V. carboxyphosphamide;
NNM, nor-nitrogen mustard.
Table 1 Details of patients studied
Patient1234567891011121314SexMFMMFMMMFMMFFFAge(yr)5350504864726437485549535652Wt(kg)6460924070707075607183738853TumorSCLC°SCLCSCLCSCLCSCLCNHLNHLNHLNHLMFHMFHBCBCUSCPdose(mg)1800100012001000120012001200120060010001200120010001200
peridol, naproxen, allopurinol, cimetidine, digoxin, potassium
chloride,cyclizine, and aluminum hydroxide.
All patients remained in the hospital for at least 25 h
post-cyclo-phosphamide administration. They collected their bulked
urine forvarious periods as follows: 1 patient 0-2, 2-8, and 8-24
h; 5 patients0-8 and 8-24 h; 8 patients 0-24 h only. Accordingly,
21 urine samplesof recorded volume were frozen at
—20°Cimmediately after collection
and then analyzed for their content of cyclophosphamide,
4-ketocyclophosphamide, carboxyphosphamide, phosphoramide mustard,
and nor-nitrogen mustard by TLC-PD.
RESULTS
The photographic record of a TLC plate which had beenvisualized
with 4-(4-nitrobenzyl)pyridine as described (27) isshown in Fig. 2.
The method clearly separates the principalurinary metabolites
4-ketocyclophosphamide, nor-nitrogenmustard, carboxyphosphamide,
and phosphoramide mustardfrom unchanged cyclophosphamide and is
specific for the derivatives of cyclophosphamide in the urine,
blank urine producing no bands (27). Each urine (typical samples
shown in lanes2-7) contained six bands of variable color intensity
and Rf 0.67(4-ketocyclophosphamide), 0.61 (cyclophosphamide),
0.40(nor-nitrogen mustard), 0.30 (unidentified metabolite,
probablythe des-2-chloroethyl compound by comparison with
authenticstandard), 0.26 (carboxyphosphamide), and 0.02
(phosphor-amide mustard). Visual inspection of the photographs of
theTLC plates, such as that shown in Fig. 2, revealed that
therelative excretion of the metabolites, in particular
carboxyphosphamide and phosphoramide mustard, was highly variable
between patients. When the black and white photographs of theTLC
plates were scanned using a densitometer, a techniquereferred to as
TLC-PD (27), chromatograms were obtainedwhich could be quantitated
for each urine specimen. From theconcentration of each metabolite
in the appropriate urine samples, the percentage of the
administered dose excreted as eachmetabolite was calculated. Table
2 gives these individual datafor each of the 14 patients together
with the means and SDs.In the 0-24-h urine after i.v. doses of
600-1800 mg cyclophosphamide, the total recovery of drug-related
material determinedby TLC-PD was 6.5-64.1% (36.2 ±17.8) dose, in
good agreement with results of workers who used radioisotopically
labeled
* SCLC, small cell lung cancer; NHL, non-Hodgkin's lymphoma;
MFH,
malignant fibrous histiocytoma; BC, breast carcinoma; US,
uterine sarcoma. t KPCPto 250 Mg/ml for cyclophosphamide,
4-ketocyclophosphamide, carboxyphosphamide, phosphoramide mustard,
and nor-nitrogen mustardin human urine and has been termed TLC-PD
(27).
Fourteen patients with various malignancies were studied, each
receiving i.v. cyclophosphamide as part of their combination
chemotherapy. The patients comprised eight males and six females,
mean age53.6 ±8.6 (SD) years, body weight 69.2 ±13.7 kg, five
with small celllung cancer, four with non-Hodgkin's lymphoma, two
with malignant
fibrous histiocytoma, two with breast carcinoma, and one with
uterinesarcoma (see Table 1). Patients received 600-1800 mg (1130
±260)cyclophosphamide with the following adjuvant drugs (number of
patients): promethazine (11), metoclopramide (10), chlorpromazine
(10),Adriamycin (9), vincristine (9), paracetamol (8),
dextropropoxyphene(8), lorazepam (8), prednisolone (6), etoposide
(6), morphine (5), ami-nophylline (2), salbutamol (2),
prochlorperazine (2), and the followingdrugs each received by one
patient only: nitrazepam, phenytoin, halo-
cx
*§__«* —. — PM12345678
Fig. 2. TLC plate which has been eluted from bottom to top and
visualizedwith 4-(4-nitrobenzyl)pyridine. Lanes 1 and 8, authentic
standards dissolved inblank urine; Lanes 2-7, urines from patients
given cyclophosphamide (CP) i.v.KP, 4-ketophosphamide; NNM,
nor-nitrogen mustard; CX, carboxyphosphamide;I'M. phosphoramide
mustard.
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DEFICIENT URINARY CARBOXYPHOSPHAMIDE EXCRETION
may be of considerable clinical importance and will be
returnedto later.
DISCUSSION
The recent development of a method which quantitates allthe
principal metabolites of the cytotoxic antineoplastic
drugcyclophosphamide has permitted a more detailed inspection ofthe
metabolic balance sheet for cyclophosphamide than hasbeen possible
hitherto. While several investigators have reported on the various
metabolites in patients, studies that haveyielded good excretion
data have invariably relied on the administration of radiolabeled
drug. The vast majority of determinations of cyclophosphamide
"metabolism" or "activation"have utilized the colorimetrie Epstein
assay (28) of total alky I•ating activity. This method, which is
usually calibrated with themost readily available metabolite
bis(2-chloroethyl)amine (nor-nitrogen mustard), can only give the
broadest of indications asto the quantitative aspects of
cyclophosphamide metabolismand no clue whatsoever as to the
qualitative pattern of metabolites in the sample analyzed, for the
simple reason that thesine qua non that each metabolite has an
identical molar extinction coefficient in the Epstein assay does
not hold. In ourhands, the molar extinction coefficients for each
authenticmetabolite differ by more than an order of magnitude.5
We
therefore sought to develop and apply a method which
wouldovercome the weaknesses of the Epstein assay to investigate
indetail the complete metabolic profile of cyclophosphamide
incancer patients.
With the exception of one breast cancer patient who excretedno
detectable nor-nitrogen mustard, all the principal metabolites of
cyclophosphamide including 4-ketocyclophosphamide,nor-nitrogen
mustard, carboxyphosphamide, phosphoramidemustard, and unchanged
drug were found in the 0 24 h urineof all 14 patients studied. The
percentage of administered doseexcreted in urine was 36.2 ±17.8%
in 0-24 h, which comparesfavorably with the findings of others who
have used radioiso-topic methods (29-31). Based upon the relative
percentage ofeach urinary metabolite (Table 2), the excretion of
each metabolite was highly variable. The two most abundant
metabolicproducts carboxyphosphamide and phosphoramide mustardhad
coefficients of variation of 118 and 51%, respectively. Alarge
proportion of this observed variation seemed to stem fromfive of
the patients who excreted 0.3% or less of the administered dose as
carboxyphosphamide (Table 2). When this wasfurther examined by
plotting the relative percentage of excretion as carboxyphosphamide
(multiplied by 10 and called thecarboxylation index, to keep the
logarithmic values positive foreach patient) as a logarithmic
distribution (Fig. 3), patientswere classified as either LC or HC.
Using the variable carboxylation index simply to phenotype patients
as either LC or HC,the origins of the metabolic carboxylation
defect were soughtfrom the data available. Firstly, the possibility
that age, sex,body weight, tumor type, or concomitant drug
treatment werethe source of the aberrant metabolism in 5 or 14
patients wasdiscounted.
The observation of a common phenotype in patients treatedwith
cyclophosphamide expressed as a marked deficiency in theexcretion
of carboxyphosphamide requires further explanation.It is not
possible that the defect lies in the initial cytochromeP-450
mediated production of the
aldophosphamide/4-hy-droxycyclophosphamide tautomerie pair, since
it would also be
5 H. L. Roberts. A. F. A. Hadidi, and J. R. Idle, manuscript in
preparation.
reflected in the excretion of other metabolites. As can be
seenfrom the metabolic scheme for cyclophosphamide given in Fig.1,
the only plausible explanation is that the defect lies in
theconversion of aldophosphamide to carboxyphosphamide byALDH (EC
1.2.1.3). Because a frank defect in this pathway hasbeen observed
it is unlikely that several enzymes or isozymescontribute to the
detoxication of aldophosphamide to carboxyphosphamide. It is
probable that a single isozymic form ofALDH which metabolizes
aldophosphamide is silent or absentin the low carboxylator
phenotype.
The formation of carboxyphosphamide from microsomalmetabolites
of cyclophosphamide has been shown to be catalyzed in vitro by ALDH
(32, 33). The selective toxicity ofcyclophosphamide against tumor
cells in vivo has been attributed to the differential tissue
distribution of ALDH (14, 33).Tissues which are high in ALDH such
as liver and kidneydeactivate cyclophosphamide effectively under
experimentalconditions (33) and in addition these organs do not
showtoxicity when the drug is administered in vivo, whereas it is
therelatively lower levels of ALDH in the tumor which permit
theconversion of circulating aldophosphamide to the
cytotoxicphosphoramide mustard by the cancer cells (34).
Moreover,the sensitivity of cells such as murine pluripotential
hemato-poietic stem cells and myeloid progenitor cells to
oxazaphos-phorine compounds can be manipulated dramatically by
thepretreatment of cells with ALDH inhibitors such as
disulfiram,diethyldithiocarbamate, and cyanamide (35).
Non-oxazaphos-phorines and acrolein showed no potentiation with
these treatments. Additionally, murine LI210 cells which have been
selected for cyclophosphamide resistance and have developed ahigh
ALDH expression (36) will revert to the wild-type sensitivity in
the presence of several ALDH inhibitors (37). Theforegoing
highlight the essential role of ALDH in determiningtarget tissue
sensitivity to cyclophosphamide.
In spite of the growing interest in the biochemistry andgenetics
of ALDH over the past decade, no deficiency in aldophosphamide to
carboxyphosphamide conversion has hithertobeen reported. In the
main, work has concentrated upon themetabolism of acetaldehyde to
acetic acid by ALDH and itspart in alcoholic disease. Human tissues
contain two mainisozymes, one with a low Km for acetaldehyde,
located in mitochondria and insensitive to disulfiram (ALDH1), and
onewith a high Km for acetaldehyde, cytosolic and inhibited
bydisulfiram (ALDH2) (38, 39). Two additional forms with a highKm
for acetaldehyde and sensitive to disulfiram (ALDH3 andALDH4) are
found in certain tissues only (39, 40). A geneticpolymorphism of
ALDH 1, associated with alcohol sensitivityand facial flushing, is
now well established. In the "unusual"
phenotype, found in 48% of Japanese, only ALDH2 is foundon
electrophoresis and thus the high affinity mitochondrialisozyme is
missing. This deficiency, found predominantly inoriental
populations, has not been described in Europeans (41).Since
cyclophosphamide metabolism is sensitive to disulfiramand other
ALDH2 inhibitors, it is highly probable that it isALDH2, ALDH3, or
ALDH4 which catalyzes the conversionof aldophosphamide to
carboxyphosphamide and was deficientin a significant proportion of
our patients. No informationregarding genetic polymorphism of
ALDH2-4 is available. Thefindings which we describe in this paper
are the first evidencefor such a polymorphism. Because of the
cytotoxic nature ofcyclophosphamide it was not possible for us to
perform familystudies to confirm the genetic transmission of LC/HC
pheno-types. However, a number of strategies at both the protein
andDNA level can be envisaged.
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DEFICIENT URINARY CARBOXYPHOSPHAMIDE EXCRETION
The detection of genetic polymorphism of the ALDH iso-
zyme responsible for the detoxication of cyclophosphamidewould
be of great clinical interest, from the point of view ofboth
tolerability of the drug and acquired resistance to it. Thelow
carboxylators who comprised 36% of our small patientcohort excreted
over twice the relative amount of cytotoxicphosphoramide mustard
and presumably therefore producedtwice the molar equivalent of the
toxic aldehyde acrolein (seeFig. 1). The TLC-PD method provides a
simple means forscreening patient populations for ascertainment of
the completemetabolic picture after cyclophosphamide administration
andthe ability to assign carboxylation phenotype. Parallel
studiesto be reported elsewhere6 with the isomer of
cyclophosphamide,ifosfamide, in non-small cell lung cancer patients
reveal asimilar proportion of patients with defective excretion of
thecarboxylic acid metabolite.
ACKNOWLEDGMENTS
The expert technical and photographic skills of Jane Baker
aregratefully acknowledged.
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