Characterization of the cytochrome P450 involved in side-chain oxidation of cyclophosphamide in humans

Article abstract of DOI:10.1007/s002280050201

Objective: Cyclophosphamide (CP) is an antineoplastic prodrug which requires bioactivation (4-hydroxylation) by the cytochrome P450 (CYP) enzymes in human liver. In parallel, P450-mediated side-chain oxidation (N-dealkylation) leads to the formation of the non-alkylating dechloroethylcyclophosphamide (DCl-CP) and chloroacetaldehyde, the latter being a potential neurotoxic agent. The enzyme responsible for side-chain oxidation has not been identified yet. We therefore used an in vitro approach to characterize the enzyme involved in N-dealkylation of CP. Methods: CP was incubated with the microsomal fraction of human liver in the presence of specific inhibitors for some P450 enzymes and in the presence of stable expressed P450 enzymes. Dechloroethylcyclophosphamide was analysed using gas chromatography and nitrogen-phosphorus detection. Results: Formation of DCl-CP increased linearly with substrate concentration over the entire concentration range (20 μmol·l^-1 to 36 mmol·l^-1). Saturation of the enzyme was not observed. Incubation with stable expressed P450 enzymes and inhibition experiments indicated that CYP 3A4 was the major enzyme involved in side-chain oxidation of CP. Conclusion: Our in vitro data indicate that side-chain oxidation of CP occurs in dose-dependent fashion in men with no saturation of this pathway even following dose escalation. Thus enhanced neurotoxicity following CP administration may result in the setting of high-dose chemotherapy. Moreover, we conclude that CP has the potential to interact with other CYP 3A4 substrates.

Full text of DOI:10.1007/s002280050201

Eur J Clin Pharmacol (1996) 51 : 297–301
© Springer-Verlag 1996
PH AR M AC OK I N E T I C S AN D D I SPOSI T I ON
F. Bohnenstengel · U. Hofmann · M. Eichelbaum · H.K. Kroemer
Characte rization of the cytochrome P450 involve d in s ide -chain oxidation of
cyclophos phamide in humans
Received: 22 January 1996 / Accepted in revised form: 27 June 1996
Abstract Objective: Cyclophosphamide (CP) is an
Introduction
antineoplastic prodrug which requires bioactivation (4-
hydroxylation) by the cytochrome P450 (CYP) enzymes
The alkylating antineoplastic drug cyclophosphamide
in human liver. In parallel, P450-mediated side-chain
is widely used in cancer chemotherapy of various malig-
oxidation (N-dealkylation) leads to the formation of
nancies. Since the parent drug shows no cytotoxicity it
the non-alkylating dechloroethylcyclophosphamide
requires bioactivation via ring oxidation by the
(
DCl-CP) and chloroacetaldehyde, the latter being a
cytochrome P450 system in the human liver to finally
yield the alkylating metabolite phosphoramide mustard
and the urotoxic metabolite acrolein. Besides this meta-
bolic activation there are some other pathways which
lead to inactive metabolites. For example, formation of
potential neurotoxic agent. The enzyme responsible for
side-chain oxidation has not been identified yet. We
therefore used an in vitro approach to characterize the
enzyme involved in N-dealkylation of CP.
Methods: CP was incubated with the microsomal frac-
tion of human liver in the presence of specific inhibitors
for some P450 enzymes and in the presence of stable
expressed P450 enzymes. Dechloroethylcyclophospha-
mide was analysed using gas chromatography and
nitrogen-phosphorus detection.
4
-ketocyclophosphamide and carboxy phosphamide by
further oxidation of the unstable intermediates
-hydroxycyclophosphamide and aldophosphamide is
4
observed [1–3]. Another inactivation pathway of CP,
the side chain oxidation (N-dealkylation), is also medi-
ated by cytochrome P450 and leads to the formation
of dechloroethylcyclophosphamide and chloroac-
etaldehyde [4]. The latter seems to be responsible for
CNS toxicity of oxazaphosphorine therapy [5]. Figure
Results: Formation of DCl-CP increased linearly with
substrate concentration over the entire concentration
9
1
91
range (20 µmol·l to 36 mmol·l ). Saturation of the
enzyme was not observed. Incubation with stable
expressed P450 enzymes and inhibition experiments
1
briefly summarizes these major pathways of CP
indicated that CYP 3A4 was the major enzyme involved metabolism.
in side-chain oxidation of CP.
Though CP metabolism has been extensively stud-
Conclusion: Our in vitro data indicate that side-chain ied over the last 25 years, many questions remain unan-
oxidation of CP occurs in dose-dependent fashion in swered. For example, little is known about the enzymes
men with no saturation of this pathway even following involved in the activation and deactivation of CP
dose escalation. Thus enhanced neurotoxicity follow- and the relative contribution of the different pathways.
ing CP administration may result in the setting of high- Only a few in vitro investigations were carried out
dose chemotherapy. Moreover, we conclude that CP to identify the P450 enzymes responsible for oxidation
has the potential to interact with other CYP 3A4 of CP. Chang et al. [6] showed that mainly CYP 2B6
substrates.
catalyses the ring oxidation of CP. These findings
appear to be questionable in view of the fact that
Key words Cyclophosphamide · CYP 3A4; side-chain 15% of Caucasian and 70% of Japanese patients do
oxidation · dechloroethylcyclophosphamide
not express CYP 2B6 at all [7]. Moreover, Yule et al.
8] showed that CP activation was affected by substrates
[
or inhibitors of CYP 3A. Activation by means of
side-chain oxidation and bioactivation of ifosfamide,
an isomer of CP, are catalysed by CYP 3A4 [6, 9]. Other
P450 enzymes play only a minor role in these reactions.
The enzyme(s) responsible for side-chain oxidation
F. Bohnenstengel · U. Hofmann · M. Eichelbaum ·
H.K. Kroemer(*)
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology,
Auerbachstrasse 112, D-70376 Stuttgart, Germany

298
Mate rials and me thods
Chemicals
All solvents used were of HPLC quality; chemicals were of analyt-
ical grade. 2-Dechloroethylifosfamide (2-DCl-IF), used as internal
standard, and DCl-CP were a generous gift from Dr. J. Pohl (Asta
Medica, Frankfurt). Cyclophosphamide monohydrate was obtained
from ICN (Meckenheim, Germany). Trizma base (TRIS[hydrox-
ymethyl]aminomethane) and Trizma -HCl (TRIS[hydroxymethyl]-
aminomethane hydrochloride) were from Sigma (Deisen hofen,
Germany).
Preparation of microsomes
Human and rat liver microsomes were prepared as described
elsewere [11]. Individual protein and P450 contents were determined
according to the method of Lowry et al. [12] and Omura and Sato
[13], respectively.
Incubation conditions for side-chain oxidation of
cyclophosphamide
Incubations were carried out at 37 °C for 30 min in the presence of
2
00 µg microsomal protein and magnesium chloride (final concen-
91
tration 6 mmol·l ) in a final volume of 250 µl. The system was
9
1
buffered with 0.05 mol·l
added as internal standard at a final concentration of 100 ng·ml
For kinetic studies increasing amounts of cyclophosphamide
TRIS buffer (pH 7.8). 2-DCl-IF was
91
.
9
1
91
(20 µmol·l to 36 mmol·l ) were added to the incubation mix-
tures. The incubation was started by adding NADPH solution (final
91
concentration 5 mmol·l ) and stopped by heating at 90 °C for
1
0 min.
Calibration samples were prepared by adding increasing amounts
of DCl-CP to mixtures which contained denaturized rat liver micro-
somes instead of human liver microsomes.
Fig. 1 Activation and inactivation pathways of cyclophosphamide
metabolism
Isolation of 2-dechloroethylifosfamide and
dechloroethylcyclophosphamide
For extraction of 2-DCl-IF and DCl-CP and separation of CP, each
8
of the microsomal incubation mixtures was transferred to a C -
cartridge (3 ml, 200 mg, Varian, Harbor City, USA) which was
conditioned with 2 ml methanol and 2 ml water. The cartridges
of CP has not been identified so far. Nevertheless
this information is pivotal for optimization of CP
therapy, as it contributes to the interaction potential were then dried by vacuum. 2-DCl-IF and DCl-CP were eluted
with 2.5 ml of a methanol-water mixture (10:90 v/v) and then
extracted with 5 ml acetic acid ethylester by shaking for 15 min.
After centrifugation the organic phase was separated and evapo-
and via formation of a neurotoxic compound to side
effects.
We therefore characterized the enzyme catalysing the
rated to dryness in a stream of nitrogen and the residue dissolved
N-dealkylation of CP using in vitro techniques. In order in 50 µl acetic acid ethylester. These extracts were analysed by gas
to obtain enzyme kinetic data and to investigate chromatography (GC).
interindividual variabilities in CP metabolism, forma-
tion of DCl-CP was analysed following incubation of
CP with the microsomal fraction of different human
Gas chromatographic determination of
dechloroethylcyclophosphamide
livers. Identification of the enzyme was achieved by (a)
correlating the rate of formation with the individual
contents of various P450 enzymes, (b) incubation of
For gas chromatographic determination of 2-DCl-IF and DCl-CP,
nitrogen-phosphorus detection was used. GC was performed in the
CP with human liver microsomes in the presence of splitless mode on a 15 m × 0.32-mm internal diameter DB-17 cap-
specific inhibitors for the individual P450 enzymes, and illary column, film thickness 0.25 µm (J&W Scientific, Fisons,
Mainz, Germany). Helium was used as carrier gas at an inlet pres-
(
c) incubation of CP with stable expressed P450
9
1
sure of 85 kPa according to a linear carrier gas velocity of 30 cm·s
enzymes. For determination of DCl-CP we used a gas
chromatographic method recently developed in our
laboratory [10].
(
120 °C). The detector temperature was 250 °C, gas flows were
91
91
91
3
ml·min
(hydrogen), 100 ml·min
(air) and 25 ml·min
(Helium, make up gas), and the bead power was set at 30 pA. The

299
9
1
initial oven temperature of 120 °C was increased by 12 °C ·min
M
Menten constant (k ) could not be calculated. Higher
to 200 °C; this temperature was held for 9 min. Retention times CP concentrations could not be tested since CP was
for 2-DCl-IF and DCl-CP were 6.5 and 7.3 min, respectively.
no longer soluble in the incubation buffer. As depicted
91
91
Calibration curves were linear from 5 ng·ml to 10 µg·ml . The
in Fig. 2,91for the concentration range up to
interassay variability averaged 4.8%.
1
4 mmol·l CP the formation of dechloroethylcy-
clophosphamide varied strongly in the different human
livers investigated.
Identification of dechloroethylcyclophosphamide formed during
the incubation by gas chromatography/mass spectrometry
The rate of formation at the concentration of
9
1
(GC/MS)
6 mmol·l
CP was correlated with the contents of
various P450 isoenzymes of six human liver prepara-
tions as shown in Table 1. Correlations were obtained
with CYP 1A2 (r = 0.928) and CYP 3A (r = 0.714) con-
tents whereas for the other isoenzymes no correlation
An HP 5985 GC /MS system was used for proving the authentic-
ity of the DCl-CP formed in the incubation experiments. The gas
chromatographic conditions were the same as described above.
Mass spectrometry was performed in the electron impact (EI) mode.
MS conditions were: transfer-line temperature 250 °C, source tem- was observed.
perature 200 °C, emission current 300 µA, electron energy 70 eV,
multiplier voltage 2800 V. The spectrum obtained after incubation
was compared to that of the authentic reference compound under
the same conditions.
Dechloroethylcyclophosphamide formation was
inhibited by ketoconazole up to 92% (6 and
91
1
4 mmol·l
CP) when the inhibitor was added at
9
1
a concentration of 50 µmol·l . At lower ketocona-
zole concentrations the inhibition was 75–82%
91
91
(
10 µmol·l ) and 61–66% (1 µmol·l ). Addition
Incubation experiments using stable expressed cytochrome P450
isoenzymes
of furafylline did not affect the rate of formation of
DCl-CP.
Incubation experiments using microsomes from human lymphoblast
cells expressing CYP 1A2 and CYP 3A4 (Gentest Corporation,
Woburn, USA) were carried out under the same conditions as the
incubations with liver microsomes. Cyclophosphamide concentra-
Incubation of cyclophosphamide with stable
expressed CYP 1A2 and CYP 3A4 resulted in forma-
tion of dechloroethylcyclophosphamide with CYP 3A4
9
1
91
9
1
91
(V = 2.70 pmol·µg ·h, 6 mmol·l
pmol·µg ·h, 14 mmol·l CP) and to a lower extent
CP; V = 8.08
tions ranged from 500 µmol·l to 14 mmol·l
.
9
1
91
9
1
91
with CYP 1A2 (V = 1.01 pmol·µg ·h, 6 mmol·l
9
1
91
CP; and V = 3.22 pmol·µg ·h, 14 mmol·l CP).
Inhibition experiments
9
1
Ketoconazole, which at a concentration of 1 µmol·l
is a selec-
tive inhibitor of CYP 3A4 [14], was incubated simultaneously with
9
1
four different concentrations of cyclophosphamide (500 µmol·l
2
,
Dis cus s ion
91
, 6 and 14 mmol·l ) under the same incubation conditions as
described above. The inhibitor was added to the incubation mix-
9
1
Here we present data indicating that CYP 3A4 is the
major enzyme responsible for side-chain oxidation of
CP. Several lines of experimental evidence support this
finding. We observed a significant correlation of CYP
tures in concentrations of 1, 10 and 50 µmol·l . CYP 1A2 was
inhibited by preincubation (10 min, 37 °C) of the microsomal mix-
91
ture with furafylline (30 µmol·l ) [15, 16].
1
A2 and 3A content in different human livers with the
Statistical analysis
Statistical analysis was performed with the InStat software from
GraphPad using Spearman’s rank correlation.
Re s ults
The authenticity of DCl-CP formed during microso-
mal incubations was proven by GC /MS. Similar reten-
tion times and mass spectra were obtained for the
analyte and an authentic reference compound. The
electron impact mass spectral data (m/z and relative
intensities) for DCl-CP were 149 (100), 56 (50), 120
(
37), 94 (15), 57 (15), 92 (13), 121 (10), 198 (5).
Formation of DCl-CP following incubation of CP
with human liver microsomes was linear in relation to
substrate concentration over the entire concentration
range (20 µmol·l to 36 mmol·l ). Therefore the
Fig. 2 Substrate dependency of the formation of dechloroethylcy-
clophosphamide following incubation of cyclophosphamide with
human liver microsomes: means with standard deviation of six liv-
9
1
91
maximum rate of formation (Vmax) and the Michaelis- ers (200 µg microsomal protein incubated for 30 min at 37 °C)

300
Table 1 Correlation of various
P450 enzyme contents versus
rate of formation (V) of
dechloroethylcyclophosphamide
following incubation of
9
P450 content (units·mg protein)
1
91
Liver
number
V at 6 mmol·l CP
9
(pmol·µg ·h91)
1
CYP 1A2 CYP 2C CYP 2D6
CYP 2E1
CYP 3A
8
12
0.2
0.1
0.7
0.0
0.0
2.8
0.8
0.9
1.2
0.5
0.3
0.1
1.3
0.5
0.8
1.2
1.7
1.1
0.3
0.4
0.3
0.3
0.2
0.2
0.3
0.9
0.7
0.5
0.4
1.2
9.94
11.4
17.5
5.81
6.60
34.5
cyclophosphamide
9
1
(
6 mmol·l ) with microsomes
1
1
1
1
3
4
6
9
of six human livers
Correlation
r = 0.928
r = 0.086 r = 90.600 r = 90.093 r = 0.714
rate of formation of DCl-CP. These data indicate trations. The resulting high k
M
leads to a low intrinsic
involvement of both enzymes in CP metabolism. clearance of this pathway, which is the confounding
Therefore both enzymes were further tested for their factor for the in vivo observation of a minor contri-
involvement in N-dealkylation of CP. Formation of bution to overall CP clearance. The linearity
DCl-CP was inhibited to more than 90% by keto- of the substrate product relationship, however, has
conazole. Recent data indicated that ketoconazole at potential clinical implications in particular in the
9
1
1
µmol·l
is a selective inhibitor of CYP 3A4 [14]. setting of high-dose therapy with CP. With high-dose
9
2
We observed 61–66% of DCl-CP formation to be inhib- CP therapy doses of up to 6000 mg·m are adminis-
ited by 1 µmol·l ketoconazole in the concentration tered [20–22]. If the enzyme for side-chain oxidation is
9
1
9
1
range of 2–14 mmol·l CP. In contrast, no inhibition not saturable, formation of the neurotoxic chloroac-
of side-chain oxidation was observed after preincuba- etaldehyde will increase in a dose-proportional fash-
tion of the microsomal fraction with the CYP 1A2 ion. Since the bioactivation pathway of CP is saturable
inhibitor furafylline [15, 16]. These data clearly indi- [6], an increase in neurotoxicity would not be paral-
cate that identification of enzymes involved in drug leled by an increase in therapeutic efficacy once a
metabolism should be carried out using different tech- threshhold dose is exceeded. These assumptions are
niques in a complementary manner. Thus the inhibi- intertwined with the observation in the present study
tion experiments clearly indicate that CYP 3A4 is the that CP side-chain oxidation shows a considerable
major enzyme involved in side-chain oxidation of CP. interindividual variability (see Fig. 2). Therefore, only
Data obtained from incubations with stable expressed a certain subset of patients with high CYP 3A4 activ-
cytochrome P450 enzymes are compatible with this ity are likely to develop neurotoxicity. Our in
hypothesis. We observed formation of DCI-CP with vitro data concerning variability of side-chain oxida-
stable expressed CYP 3A4 and CYP 1A2. The rate tion of CP are in good agreement with in vivo results
of formation, however, was considerably higher for [18].
9
1
91
CYP 3A4 (2.70:1.01 pmol·µg ·h at 6 mmol·l
8
3
;
In summary we conclude that CYP 3A4 is the major
9
1
91
.08:3.22 pmol·µg ·h at 14 mmol·l CP for CYP enzyme responsible for side-chain oxidation of CP. The
A4:CYP 1A2). Moreover, the natural abundance of enzyme is not saturable over the concentration range
CYP 1A2 in human liver is at most one-third of that from 20 µmol·l to 36 mmol·l CP. These findings
of CYP 3A4 [7]. Taking these data into account fur- have potential implications for both interaction poten-
ther supports the hypothesis that CYP 1A2 contributes tial and side effects of CP.
9
1
91
only to a minor extent to side-chain oxidation of CP.
CYP 3A is a major enzyme in the metabolism of a Acknowledgements This work was supported by the Dr. Mildred
Scheel Foundation (Bonn, Germany), Grant No. W90/92/Ei2, and
the Robert Bosch Foundation (Stuttgart, Germany). The authors
are grateful to S. Johnson for technical assistance.
variety of drugs including other cytostatics such as
etoposide [17]. The latter drug is often used in combi-
nation with CP, giving rise to the potential of a
drug/drug interaction. Since the side-chain oxidation
contributes only to a minor extent to overall clearance
of CP [18], such interactions are unlikely to change the Re fe re nce s
disposition of CP itself but rather that of the other
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