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Vol. 27, Issue 3, 417-421, March 1999
Department of Hematology, Yorkhill NHS Trust, Glasgow (S.M.Y.); Departments of Pharmacological Sciences (S.M.Y., D.W., L.M.S., S.C., A.K.D.), Child Health (S.M.Y., M.C., A.D.J.P.), Statistics (M.C.), and the Cancer Research Unit (A.V.B.), The University of Newcastle upon Tyne, United Kingdom
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Fluconazole is increasingly used in children receiving chemotherapy. Many of these patients are being treated with cyclophosphamide, which must undergo hepatic metabolism to produce active alkylating species. As a consequence of the cytochrome P-450 inhibitory properties of fluconazole, a potential interaction exists between these two agents that could influence the therapeutic effect of cyclophosphamide. To investigate this interaction, a retrospective case series of patients was chosen from a population of children with a previously established profile of cyclophosphamide metabolism. Twenty-two children who were not receiving other therapy known to influence drug metabolism were selected and analyzed in terms of fluconazole treatment; of these, nine were receiving fluconazole and thirteen were identified as controls. Study design was not randomized. The plasma clearance of cyclophosphamide was lower in patients receiving fluconazole [mean(SD) 2.4(0.71) versus 4.2(1.2) l/h/m2, p = .001]. In vitro studies were performed to characterize the interaction between fluconazole and cyclophosphamide in six human liver microsomes. The concentration of fluconazole required to reduce the production of 4-hydroxycyclophosphamide to 50% of control values (IC50) varied between 9 and 80 µM (median 38 µM). Further studies of the effect of fluconazole on 4-hydroxycyclophosphamide production in vivo are warranted to determine whether this interaction reduces the therapeutic effect of cyclophosphamide in clinical practice.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cyclophosphamide is widely used in the treatment
of cancer in children. It is a prodrug that must be metabolized by
cytochrome P-450 enzymes
(P-450s)1 to
produce alkylating species, which are responsible for
the drug's cytotoxic effects (Sladek, 1988
). The extent of
cyclophosphamide elimination has been shown to correlate with both
treatment efficacy and toxicity (Ayash et al., 1992). Previous studies
have reported a high degree of interpatient variation in
cyclophosphamide metabolism in children as a result of inherited and
environmental differences in enzyme expression. It is likely that some
of this variation results from the effects of concurrent drug therapy
upon the activity of the P-450s involved (Tasso et al., 1992; Yule et
al.,1995).
Cyclophosphamide undergoes extensive metabolism to produce alkylating
species and several inactive byproducts. The initial step is
hydroxylation of the oxazaphosphorine ring to produce 4-hydroxycyclophosphamide, which exists in equilibrium with its tautomer aldophosphamide. Further breakdown of aldophosphamide by
spontaneous 
-elimination releases phosphoramide mustard, the ultimate alkylating species. Alternatively, aldophosphamide may be
oxidized to inactive carboxyphosphamide (CX). The other principal inactivation product of cyclophosphamide is
dechloroethylcyclophosphamide (DCCP), which is produced as a result of
a P-450-dependent oxidative N-dealkylation reaction (Sladek,
1988; Yule et al., 1995). The individual P-450s capable of catalyzing
the hydroxylation of cyclophosphamide by human liver microsomes were
initially identified as CYP2A6, CYP2B6, CYP2C8, CYP2C9, and CYP3A4
(Chang et al., 1993). A subsequent study using P-450 inhibitors
demonstrated that, of these, the role of CYP2C9 and CYP3A4 was most
significant (Ren et al., 1997).
In addition to the formation of phosphoramide mustard, -elimination
of aldophosphamide produces an equimolar quantity of acrolein, which
has been implicated in the etiology of hemorrhagic cystitis (Cox,
1979). In clinical practice, hemorrhagic cystitis is prevented by
simultaneous administration of sodium 2-mercaptoethane sulfonate
(mesna), which undergoes extensive oxidation to dimesna (dithio-bis-mercaptoethanesulphonate) in the blood stream.
This reaction is reversed in renal tubular cells, which allow mesna to
be secreted into the urine where it binds with and inactivates acrolein
(Shaw and Graham, 1987; Hilgard and Pohl, 1990). Hemorrhagic cystitis
is most common after high-dose cyclophosphamide therapy, and mesna is
not routinely given to patients receiving doses of less than 1 g/m2.
Fluconazole is a triazole antifungal that is increasingly used in
children receiving chemotherapy or undergoing bone marrow transplantation as prophylaxis against infections with Candida sp (Grant and Clissold, 1990; Slavin et al., 1995). Several
investigators have reported that fluconazole inhibits the metabolism of
drugs that are substrates of CYP2C8, CYP2C9 or CYP3A4, (Blum et al., 1991; Jurima-Romet et al., 1994; Schwartz et al., 1995; Kunze et al.,
1996; Osowski et al., 1996; Trapnell et al., 1996; Varhe et al., 1996).
Thus, a potential interaction exists between fluconazole and
cyclophosphamide that may reduce the therapeutic efficacy of
cyclophosphamide. This investigation describes the effect of fluconazole on the pharmacokinetics and metabolism of cyclophosphamide in children and characterizes the nature of this interaction in six
human liver microsomes.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Clinical Studies. Twenty-two children (8 females) aged between 2 months and 18 years (median 4 years) were investigated. No patient at the time of study, or in the 28 days before the study, had received other drugs known to interfere with P-450 activity. All patients were treated with cyclophosphamide as a constant rate 1-h infusion; the median dose administered was 1050 mg/m2 (range 270-2130 mg/m2). Nine children were receiving fluconazole (5 mg/kg once daily, either orally or as a 1-h i.v. infusion) for at least 1 week before the study (Table 1). Children being treated with fluconazole received a higher dose of cyclophosphamide (median 1285 mg/m2 versus 600 mg/m2), and as a result, were more likely to receive mesna during chemotherapy.
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20°C
before analysis. The concentrations of cyclophosphamide, CX, and DCCP
in plasma were determined using a high performance thin layer
chromatography-photographic densitometry assay, which has been
described previously (Yule et al., 1995). Unfortunately, this technique
does not reliably detect phosphoramide mustard, which requires bedside
derivatization to increase its stability. Although several methods for
measuring phosphoramide mustard have been described, none have proven
to be reproducible (Yule et al., 1993). Cyclophosphamide
pharmacokinetics were assumed to follow a one-compartment open model
with first order elimination kinetics assuming constant rate drug
infusion. Estimates of cyclophosphamide pharmacokinetic parameters were
obtained by maximum likelihood using ADAPT II (D'Argenio and
Schumitzky, 1992). The variance was weighted in favor of
1/C2, where C was the absolute concentration of
cyclophosphamide in plasma. To allow for heteroscedasticity, the error
variance at time, o2(t), was assumed to take the
functional form o2(t) = (g + dm(t))2, where m(t) was the expected, or fitted,
plasma concentration at time t. The parameters g and d were estimated
as part of the fitting process and were not specified a priori. The
area under the concentration-time curve (AUC) for individual
metabolites was calculated using the trapezoidal rule. Comparisons
between children receiving fluconazole and the remainder of the study population were performed using Student's unpaired t test.
Written consent from the child's parents and, where appropriate, from the subjects themselves, was obtained before participation in the
study. The project was approved by the joint ethical committee of the
Medical School of the University of Newcastle upon Tyne and the Royal
Victoria Infirmary, Newcastle upon Tyne.
Human Liver Microsomal Incubations.
Human liver microsomes were purchased from the Keystone Skin Bank
(Exton, PA and The International Institute for the Advancement of
Medicine, University of Leicester, UK). Each batch of microsomes had
been characterized for the activity of several P-450s by the suppliers;
CYP2A6 activity was determined by the rate of coumarin 7-hydroxylation,
CYP2C19 activity was determined as the rate of mephenytoin
4-hydroxylation, and CYP3A activity was determined by the rate of
production of
[14C]6-hydroxytestosterone. Several
additional studies were performed on these human liver microsomes in
our laboratory as part of other ongoing studies. CYP2C9 activity was
determined as the rate of diclofenac 4-hydroxylation (Leeman et al.,
1993) and the expression of CYP2C8 and CYP3A4 was measured using
immunoblotting with enhanced chemiluminescence detection
(Amersham International, Amersham, UK) using anti-CYP2C (Dr. P. Beaune,
Centre Universitaire des Saints-Pères, Paris, France) and
anti-CYP3A antibodies (Dr. J. Hardwick, Northeastern Ohio University,
Rootstown, OH).
). Microsomes were incubated at 0.25 to 0.5 mg/ml protein for 45 min in conditions described previously (Walker et al., 1994).
Fluconazole was added to individual microfuge tubes to provide
concentrations of 5, 10, 50, 100, 250, and 500 µM and incubated at
37°C for 10 min before the initiation of the reaction by the addition
of cyclophosphamide to provide a final substrate concentration of 1 mM.
This concentration was selected to reflect in vivo levels of
cyclophosphamide in pediatric patients (Juma et al., 1984; Tasso et
al., 1992). All measurements were the result of duplicate observations.
Experiments were performed on six different microsomes to determine the
concentration of fluconazole at which 4-hydroxycyclophosphamide
production was reduced to 50% of control values
(IC50). Estimates of IC50
were made by fitting a Hill equation to data for
4-hydroxycyclophosphamide formation as a function of fluconazole
concentration using GraphPad Prism (GraphPad Software, Inc., San Diego,
CA). The initial value was fixed at 100% and the Hill coefficient to
1. To investigate whether the presence of mesna altered the extent of
cyclophosphamide 4-hydroxylation, a series of microsomal incubations
were performed with and without mesna at a concentration of 1 mM.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Clinical Studies. The disappearance of cyclophosphamide from plasma was monoexponential in all cases. Details of the pharmacokinetic parameters and of individual metabolite production in both groups are presented in Table 2. Cyclophosphamide Cl in children receiving fluconazole was lower than that in controls (2.4(0.71) versus 4.2(1.2) l/h/m2; p = .001; Fig. 1.). This difference persisted despite using the analysis of covariance to correct for any possible effect of administered dose or patient age on Cl (p = .02 and p = .007, respectively). Children receiving fluconazole also exhibited a longer half-life (6.7(4.9) versus 2.2 (0.49) h; p = .003). There was no accompanying difference in volume of distribution. Fluconazole did not influence the production of either CX or DCCP.
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Human Liver Microsomal Incubations. The rate of 4-hydroxycyclophosphamide production was linear during the 45-min incubation period and was not affected by the presence of mesna (data not presented). The median IC50 of fluconazole was 38 µM (range 9-80 µM; Fig. 2.). No correlation was seen between the extent of inhibition and CYP2A6, CYP2C, CYP2C9, or CYP3A4 activity. Similarly, there was no correlation between the expression of either CYP2C8 or CYP3A4 as measured by immunoblotting and the inhibition of cyclophosphamide 4-hydroxylation.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The results of this investigation suggest that treatment with
fluconazole may inhibit the P-450s involved in cyclophosphamide metabolism. In vivo studies demonstrate a lower plasma Cl of
cyclophosphamide in children receiving fluconazole. Although the
two groups of patients studied were heterogenous in respect of their
clinical condition, prior cytotoxic therapy, and accompanying
medication, these variables were unlikely to influence the results in
the systematic fashion observed here. Previous studies have
demonstrated that the pharmacokinetics of cyclophosphamide remain
linear across the doses administered in this investigation; thus,
differences between the two groups of children are unlikely to result
from saturation of elimination pathway(s) (Wilkinson et al., 1983; Busse et al., 1997). Further confirmation that the observed effect of
fluconazole on the Cl of cyclophosphamide was not solely the result of
variation in administered dose was provided using the analysis of covariance.
Because of the relationship between cyclophosphamide dose and
hemorrhagic cystitis the use of mesna as a uroprotectant occurred almost exclusively in children receiving fluconazole. Children receiving mesna concurrently with cyclophosphamide were treated with
equivalent doses of the two drugs (James et al., 1987). Our study found
neither evidence of any effect of mesna upon the pharmacokinetics of
cyclophosphamide nor interference with 4-hydroxycyclophosphamide production by human liver microsomes upon the addition of mesna. Although mesna forms reversible adducts with circulating
4-hydroxycyclophosphamide, it does not influence the elimination
kinetics of cyclophosphamide itself (Shaw and Graham, 1987). Thus it is
unlikely that preferential administration of mesna contributed to the
observed differences in cyclophosphamide metabolism.
Several patients in both study groups were receiving treatment with
oral trimethoprim-sulfamethoxazole (240-480 mg twice daily, administered 3 days per week) as prophylaxis against respiratory infections. Previous reports have demonstrated that
trimethoprim-sulfamethoxazole potentiates the clinical effects of
S-warfarin and tolbutamide, probably by reducing the
activity of CYP2C9 (O'Reilly, 1980; Wing and Miners, 1985; Kunze et
al., 1996). We found no significant effect of
trimethoprim-sulfamethoxazole on cyclophosphamide metabolism in
this study. This may reflect the relatively small doses of the
antibiotic administered in this setting.
At this time it is uncertain whether the 4-hydroxylation and
N-dechloroethylation of cyclophosphamide are catalyzed by
identical P-450s in vivo. Distinct isoforms are responsible for the
production of these metabolites in animals (Yu and Waxman, 1996). DCCP
production by human liver microsomes is dependent upon CYP3A4 activity
and relatively unaffected by any contribution from the CYP2C family (Ren et al., 1997). The absence of any effect of fluconazole on the
production of DCCP in this study also suggests that these reactions are
catalyzed by different P-450s. This result should be interpreted with
caution because of the difficulty in measuring plasma concentrations of
DCCP in vivo. Circulating concentrations of this metabolite were below
the lower limit of detection in 44% of children receiving fluconazole.
As expected, the production of CX from aldophosphamide by aldehyde
dehydrogenase was unaffected by fluconazole (Dockham et al., 1992).
Fluconazole is thought to prevent the replication of C sp by
binding to the haem moiety of fungal CYP450 liters1A1 (Lanosterol 14 
-demethylase), thereby reducing the production of ergosterol, an
essential component of the fungal cell wall (Hitchcock et al., 1990).
Although a less potent inhibitor of CYP450 enzymes than imidazole
antifungals such as ketoconazole (Ervine et al., 1996), fluconazole
inhibits the metabolism of several CYP2C and CYP3A4 substrates in
humans. It is likely that irreversible binding of the triazole ring
structure of fluconazole to P-450s from the CYP3A family, and
particularly CYP2C9, is responsible for the reduction in
cyclophosphamide Cl observed in this study (Kunze et al., 1996).
Two studies have described the pharmacokinetics of fluconazole in
children. The first of these reported peak plasma concentrations of 20 to 30 µM after an initial dose of 4 mg/kg. Steady state concentrations, achieved after 4 days of continuous therapy, were approximately 1.6 times greater than the initial peak value (Lee et
al., 1992). A second pediatric study reported peak concentrations of 30 to 60 µM after a single dose of 6 mg/kg delivered as a 1-h infusion
(Seay et al., 1995). It is likely that only the unbound proportion of
circulating fluconazole is available to interact with P-450s in vivo.
The plasma-protein binding of fluconazole is low, representing only
11% of total plasma concentrations (Debruyne and Ryckelynck, 1993).
Thus, the peak free plasma concentration of fluconazole obtained during
the present study lies between 20 and 50 µM. These concentrations are
of a similar order to the IC50 values obtained
from human liver microsomal studies (9-80 mM). The similarity between
these values suggests that significant inhibition of cyclophosphamide
metabolism by fluconazole may occur in vivo. Such an effect may be
particularly marked in patients with pre-existing renal impairment in
whom peak fluconazole concentrations are greater (Lee et al., 1992).
In this study IC50s were not directly related to the activity of individual P-450s. The absence of a direct correlation may result from the incomplete characterization of the human liver microsomes used or the small number of microsomes tested, but is more likely to reflect the complexity of cyclophosphamide metabolism in humans. Inhibition of a single or a small number of P-450s by fluconazole is likely to have different effects in different microsomes depending upon the level of expression of the affected isoforms and of other unaffected P-450s capable of catalyzing the 4-hydroxylation of cyclophosphamide. Such variation obscured any correlation between the extent of inhibition and the expression of individual P-450s in this study.
We were unable to compare the clinical consequences of the inhibition of cyclophosphamide metabolism by fluconazole in terms of drug toxicity and treatment efficacy because of differences in the underlying malignancies within the study population. Such an analysis is also complicated by the widespread use of multiagent chemotherapy regimens. The conclusions of our investigation are weakened by the fact that they are derived from a retrospective series of patients rather than a randomized study. Further difficulties in the interpretation of our results are provided by the use of a wide range of doses of cyclophosphamide in addition to many other drugs given concurrently. This situation, however, accurately reflects the nature of pediatric oncology patients making pure randomized controlled studies difficult to perform.
Because of the complex nature of cyclophosphamide metabolism the clinical consequences of a reduction in clearance is uncertain. Our results illustrate the need for further studies of the effect of fluconazole on 4-hydroxycyclophosphamide production in vivo to determine whether this interaction reduces the therapeutic effect of cyclophosphamide in clinical practice
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Footnotes |
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Received May 26, 1998; accepted November 16, 1998.
This work was supported by the The Tyneside Leukaemia Research Fund and the North of England Childrens Cancer Research Fund.
Send reprint requests to: Dr. S. M. Yule, Department of Haematology, Yorkhill NHS Trust, Glasgow G3 8SJ, United Kingdom.
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Abbreviations |
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Abbreviations used are: P-450s, cytochrome P-450 enzymes; DCCP, dechloroethylcyclophosphamide; CX, carboxyphosphamide; mesna sodium, 2-mercaptoethane sulphonate; AUC, area under the concentration-time curve.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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