Download Af,A^yV"-Triethylenethiophosphoramide (Thio

[CANCER RESEARCH 51, 2340-2345, May 1, 1991]
Af,A^yV"-Triethylenethiophosphoramide
(Thio-TEPA)
Oxygénationby Constitutive
Hepatic P450 Enzymes and Modulation of Drug Metabolism and Clearance in
Vivo by P450-inducing Agents1
Sze-fong Ng and David J. Waxman2
Department of Biological Chemistry and Molecular Pharmacology and Dana-Farber Cancer institute, Harvard Medical School, Boston, Massachusetts 02115
ABSTRACT
ever, their effectiveness generally lies in their ability to disrupt
key cellular processes (e.g., DNA replication) in rapidly prolif
erating cells (1). Several alkylating agent anticancer drugs also
interact with a group of membrane-bound monooxygenase en
zymes termed cytochromes P450 (2). The P450 enzymes are
expressed at high levels in the liver, where they play a key role
in the oxidative metabolism of a diverse group of endogenous
substrates as well as foreign compounds including many drugs
and carcinogens (3, 4). Several anticancer drugs, such as cyclophosphamide (5, 6) and procarbazine (7), are activated and/or
inactivated in the liver by P450-dependent metabolic pathways.
The thiophosphoramidate
alkylating agent thio-TEPA3 is
used for treatment of bladder and meningea! carcinomas and
for metastatic carcinoma of the breast, particularly in patients
with refractory malignancies, where high dose therapy protocols
can be applied in association with autologous bone marrow
transplantation (8). Recent studies on the metabolism of thioTEPA have revealed that its major route of biotransformation
involves oxidative desulfuration to yield TEPA, an active me
tabolite (9, 10). This reaction is cytochrome P450 dependent
and is actively catalyzed by a phenobarbital-inducible rat liver
substrate), while several other constitutive hepatic P450s exhibited sig
P450 designated UBI (form PB-4)4 (13). TEPA production is
nificantly lower or undetectable activities (turnover, <0.15 min' P450'1).
associated with a mechanism-based (suicidal) inactivation of
Metabolism of thio-TEPA by purified P450 IK 11 was associated with a
the P450 by reactive metabolite(s) generated during the desul
time-dependent inactivation of the cytochrome analogous to that previ
furation process (13). These observations are consistent with
ously shown to accompany thio-TEPA metabolism catalyzed by P450
other studies that point to the involvement of hepatic enzymes
UBI. Depletion of hepatic P450 IK 11 by cisplatin treatment of adult
(probably P450s) in the conversion of thio-TEPA to metabolites
male rats led to a 70% reduction of TEPA formation catalyzed by the
isolated liver microsomes, suggesting that cisplatin may influence thiothat retain or perhaps have enhanced alkylation activity (9, 10,
14). Although an important role for the phenobarbital-inducible
TEPA pharmacokinetics when these two drugs are given in combination.
The extent to which hepatic P450s contribute to thio-TEPA metabolism
P450 UBI in thio-TEPA metabolism has been established (13),
and clearance in vivo was assessed by monitoring thio-TEPA and TEPA
little is known about the capacity of constitutively expressed
pharmacokinetics in rats that exhibit widely differing rates of microsomal
hepatic P450 enzymes to oxygenate this drug. Uninduced rat
thio-TEPA metabolism, i.e., uninduced female and male rats, and male
liver does not contain significant levels of UBI and, overall,
rats treated with the P450 UBI inducers clofibrate and phénobarbital.
exhibits
a markedly different profile of P450 enzymes when
In accord with the microsomal activities, conversion of thio-TEPA to
compared to drug-induced liver (11). Studies described in this
TEPA was less extensive and thio-TEPA elimination slower in female
report serve to identify and characterize two constitutively
than in male rats. Clofibrate and phénobarbitalboth accelerated thioexpressed P450 enzymes that play a major role in thio-TEPA
TEPA clearance by increasing metabolism to TEPA; this was evidenced
metabolism in uninduced liver tissue.
by a 4-fold increase in the peak plasma level of the oxo-metabolite in the
inducer-pretreated animals. These findings demonstrate that liver P450
Among the many possible contributions to variability in the
enzymes have a major impact on thio-TEPA metabolism and clearance
efficacy and toxicity of anticancer drugs, a major factor is the
and further suggest ways in which thio-TEPA pharmacokinetics might
bioavailability of cytotoxic molecular species (15). Although
be modulated in vivo to achieve improved therapeutic effects.
this pharmacokinetic parameter is influenced by physiological
variables, such as renal clearance, metabolic parameters, includ
ing hepatic enzyme-catalyzed drug activation and detoxifica
INTRODUCTION
tion, can also play a critical role. In the case of thio-TEPA,
both
the parent drug and its major circulating oxo-metabolite,
Cytotoxic drugs are widely used in the treatment of cancer.
The therapeutic modes of action of these chemicals vary; how- TEPA, possess significant antitumor activity. Pharmacokinetic
The cancer chemotherapeutic drug A',,V',.V"-trieth>lenethiuphosphoramide (thio-TEPA) is oxidatively desulfurated to yield the active metab
olite A'.W.yV-triethylenephosphoramide (TEPA) in a reaction catalyzed
by the phenobarbital-inducible rat liver P450 enzyme UBI. In the current
study, the role of constitutively expressed P450 enzymes in thio-TEPA
metabolism was studied using purified P450s, isolated liver microsomes,
and intact rats. Metabolism of thio-TEPA (100 MM)to TEPA by uninduced adult female and male rat liver microsomes proceeded at initial
rates of 0.10 and 0.28 nmol TEPA formed/min/mg microsomal protein,
respectively. Although these rates are low compared to those catalyzed
by phenobarbital-induced liver microsomes (3.5 nmol TEPA/min/mg),
they are sufficient to contribute to the systemic metabolism of this drug.
Thio-TEPA metabolism catalyzed by uninduced female liver microsomes
was -70% inhibitable by antibodies selectively reactive with P450 11(6.
For the uninduced male liver microsomes, which exhibit a severalfold
higher rate of thio-TEPA metabolism, enzyme activity was only 15-20%
inhibitable by these antibodies but was 80-85% inhibited by an antiP450 IIC6 monoclonal antibody cross-reactive with P450 IK II, which
is expressed only in the males. Consistent with these observations,
purified P450s IK 11 and IIC6 both oxidized thio-TEPA in reconstituted
systems (turnover, 1.1 and 0.3 min'1 P450 ', respectively, at 100 MM
Received11/26/90;accepted2/22/91.
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 in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported in part by Grant CN-14 from the American Cancer Society
[D. J. W.J.
1To whom correspondence should be addressed, at Dana-Farber Cancer Insti
tute, JF-525, 44 Binney Street, Boston, MA 02115.
3 The abbreviations used are: thio-TEPA, yv,A'',jV"-triethylenethiophosphoramide; TEPA, JV,iV',A'"-triethylenephosphoramide; P-450, cytochrome P450; tw,
half-life.
* Rat liver P4SO protein designations used in earlier studies from this and
other laboratories are summarized elsewhere (11). Systematic P450 gene product
designations (12) are indicated by Roman numerals. Specifically, the P4SOs
currently designated UBI, IIC6, and IIC11 correspond to forms called PB-4, PBI, and 2c in our earlier reports (6, 13), as noted in Table 1 of this study.
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MODULATION
OF HEPATIC THIO-TEPA METABOLISM
studies in patients have demonstrated that total body clearance
of TEPA is significantly slower than that of the parent drug
(16, 17). Moreover, there is evidence for the saturability of
TEPA production at clinically relevant doses (17). These and
other observations indicate that it may be feasible to modulate
the activity of thio-TEPA through alterations in its pharmacokinetics. Studies described in this report demonstrate that thioTEPA metabolism and clearance can, indeed, be modulated
through the application of P450 form-specific inducing agents
that alter hepatic monooxygenase activity toward this chemotherapeutic drug substrate.
MATERIALS
AND METHODS
Materials. Thio-TEPA, TEPA, other chemicals and reagents, and
polyclonal and monoclonal anti-P450 antibodies were those described
previously (13). Liver microsomes isolated from uninduced and phenobarbital-induced Fischer 344 rats were prepared by a calcium precip
itation method (18) and P450 enzymes were purified to apparent
homogeneity from Sprague-Dawley rats as summarized elsewhere (19).
Purified P450 IIC7 was kindly provided by Dr. C. R. Wolfe, Imperial
Cancer Research Fund, Edinburgh, United Kingdom. Anti-P450 anti
bodies used in the antibody inhibition experiments were purified from
polyclonal antisera or ascites fluid containing mouse monoclonal IgG
or IgM (20).
Thio-TEPA Metabolism Assays. Metabolism of thio-TEPA to TEPA
catalyzed by liver microsomes or purified, reconstituted P450 enzymes
and product analysis with quantitation by gas chromatography were
carried out as detailed previously ( 13), with the following modifications:
enzyme reactions were scaled down to a total volume of 0.05-0.20 ml;
and the microsomal protein concentration increased to 0.9 mg/ml.
Thio-TEPA concentrations ranged from 0.1 to 1 mM and total reaction
times from 5 to 90 min as indicated in the text. Enzyme activities
(initial rates) were estimated from the initial, linear portion of reaction
curves such as those shown in Fig. 1 (see below). Deviations from
linearity may reflect enzyme inactivation that results from thio-TEPA
metabolism (13). Antibody inhibition experiments were performed as
described previously (13), with complete reaction mixtures (minus thioTEPA and NADPH) preincubated with anti-P450 IgG or control
PB. Moie
•UT, Mote
O UT. Femoie
PB, Male
UT, Male
UT. Femole
TIME (min)
Fig.
Thio-TEPA
metabolism catalyzed(P'li)
by uninduced
untreated)
adult
male
andI. female
and phenobarbital-induced
adult male (IT,
rat liver
microsomes.
Isolated liver microsomes were incubated at 37°Cwith thio-TEPA, 0.1 mM (A)
or 1.0 mM (B), and 1 mM NADPH for the indicated times. Aliquots were then
removed for gas chromatography analysis of TEPA as described under "Materials
and Methods."
(nonimmune) IgG for 30 min at 20-22°C,followed by the addition of
thio-TEPA and NADPH to initiate the oxygénationreaction.
Thio-TEPA Plasma Pharmacokinetics. Adult male and female
Fischer 344 rats (9-12 weeks of age) were used to minimize interanimal
differences in P450 enzyme levels and activities, which appear to be
more prominent in outbred strains such as Sprague-Dawley (21). Rats
were untreated or were treated with either clofibrate (daily injection of
40 mg/100 g body weight for 3 consecutive days, i.p. in 0.5 ml corn
oil) or sodium phénobarbital(daily injection of 8 mg/100 g body weight
for 4 consecutive days, i.p. in 1 ml 0.9% NaCl, pH 9.0) to induce
hepatic P450 UBI (6) prior to the start of the experiment. Thio-TEPA
was freshly dissolved in double-distilled water at 8 mg/ml and then
injected into the tail vein at 2 mg/100 g body weight. Individual rats
were sacrificed after brief asphyxiation under CO: at time points
ranging from 5 to 240 min after drug administration. Blood samples
(~1 ml) were immediately withdrawn from the heart and placed in icecold microcentrifuge tubes containing 100 USP units of heparin. Ali
quots of the heparinized blood (0.01 ml) were mixed with 0.4 ml ethyl
acetate containing diphenylamine (5 /AI) as internal standard, and 1 /il
of the resultant mixture was then analyzed for plasma thio-TEPA and
TEPA levels by gas chromatography (13). Plasma samples could be
kept frozen at -80°C for at least 6 months without detectable changes
in thio-TEPA and TEPA levels.
RESULTS
Metabolism of Thio-TEPA to TEPA Catalyzed by Uninduced
Rat Liver Microsomes. Thio-TEPA metabolism catalyzed by
liver microsomes isolated from uninduced adult male and fe
male rats was examined, as shown in Fig. 1. TEPA, the only
metabolite detected in these incubations, was formed by the
male liver microsomes at initial rates (0.28 and 1.33 nmol/
min/mg microsomal protein at 0.1 and 1.0 mM thio-TEPA,
respectively) that were severalfold greater than those catalyzed
by adult female rat liver .nicrosomes (0.10 and 0.23 nmol/min/
mg at 0.1 and 1.0 mM thio-TEPA, respectively) (Fig. 1). Al
though these rates are much lower than those catalyzed by
phenobarbital-induced microsomes (cf., 3.5 and 5.6 nmol TEPA
formed/min/mg microsome at 0.1 and 1.0 mM thio-TEPA,
respectively), they appear to be high enough to contribute to
systemic metabolism of this drug. Thus, a yield of 200-mg liver
microsomal protein/10-g liver/200-g rat suggests that an un
induced male rat liver could metabolize thio-TEPA at a rate of
3.4 Ã-/mol/h.This can be compared to a therapeutic dose of thioTEPA of 5-20 /jmol (1-4 mg) per 200-g rat.
Selective Inhibition of Microsomal Thio-TEPA Metabolism
by P450 Form-Specific Antibodies. Although UBI is the major
catalyst of thio-TEPA metabolism in phenobarbital-induced rat
liver (13), this P450 is virtually absent from uninduced liver
and hence is unlikely to be the predominant catalyst of thioTEPA metabolism in uninduced liver microsomes. This was
confirmed by the inability of anti-IIBl antibodies to inhibit
TEPA formation catalyzed by uninduced liver microsomes un
der conditions where they do inhibit by >90% the correspond
ing reaction catalyzed by phenobarbital-induced liver micro
somes (13). In order to identify the P450 enzyme(s) that partic
ipate in the biotransformation of thio-TEPA in uninduced rats,
inhibitory antibodies reactive with several other forms of P450
were used as probes in the microsomal assays. With uninduced
adult male rat liver microsomes, a monoclonal antibody prep
aration reactive with both P450 IIC11 (form 2c) and P450IIC6
(form PB-1)4 (22) inhibited this microsomal activity by 80-85%
(Fig. 2A), whereas polyclonal anti-IIC6 antibodies that are more
specific for IIC6 (23) inhibited this activity by only 15-20%
(Fig. 2B). Little or no inhibition (<10%) was effected by non-
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MODULATION
A. Anti-NCI
OF HEPATIC THIO-TEPA
1/IIC6
005
0.10
0 15
0.20
0 25
Anti-P450 IIC11/IIC6 MAb (ug IgM/ug microsome)
'
-
B. Anti-IIC6
0.5
1.0
1.5
20
2-5
Anti-P450 IIC6 Antibodies fug IgG/ug microsomei
Fig. 2. Inhibition of microsomal thio-TEPA metabolism by P450 form-specific
antibodies. Liver microsomes (90 ¿ig)isolated from uninduced adult male and
female rats were preincubated, in a final volume of 85 //I. with varying amounts
of purified monoclonal antibody reactive with P450 IIC11 and P450 IIC6 \MAb
DI (22)} (A), or polyclonal antibodies selectively inhibitory toward P450 IIC6 (B)
for 30 min at 20'C. After the addition of thio-TEPA (1.0 mivi), the incubations
were transferred to 37'C and NADPH (1 ITIM)was added to initiate the monooxygenase reaction. Aliquots of the reaction mixtures were removed 15 min later
for gas chromatography analysis as described under "Materials and Methods."
Control incubations to monitor the effects of nonspecific IgG are shown for both
male (•)and female (A) liver microsomes. Similar results were obtained when
the experiments were performed at a thio-TEPA concentration of 0.1 mM.
specific IgG or by antibodies reactive with several other consti
tutive or inducible forms of P450 (i.e., P450s IIIA1, IIIA2,
HAI, UBI, IIB2, lAl, and IA2; data not shown). Together,
these data establish that IIC11, and to a lesser extent IIC6,
make important contributions to thio-TEPA metabolism cata
lyzed by uninduced adult male rat liver microsomes.
While IIC6 is expressed in liver at similar levels in adult rats
of both sexes, the expression of IIC11 in rat liver is male
specific (11). When thio-TEPA metabolism catalyzed by unin
duced female microsomes was probed with either the monoclo
nal antibody, reactive with both IIC11 and IIC6, or the poly
clonal anti-IIC6 antibodies, thio-TEPA metabolism was inhib
ited by up to 70% (Fig. 2). Thus, in female rat liver microsomes,
IIC6 alone plays a major role in thio-TEPA metabolism. Al
though polyclonal anti-IIC6 antibodies can cross-react with
P450s IIC13, IIC7, and IIC12 (e.g., 23,24), the Fischer rats
used in these experiments do not contain detectable P450IIC13
protein (21). P450s IIC7 and IIC12 are also unlikely to con
tribute significantly to thio-TEPA metabolism in the female
liver microsomes because in microsomes isolated from hypophysectomized rats, in which IIC7 and IIC12 are both depleted
by more than 90% (e.g., Refs. 25 and 26), thio-TEPA metabo
lism is not decreased but rather is moderately increased, as is
IIC6 (data not shown).
Thio-TEPA Metabolism Catalyzed by Purified, Reconstituted
Rat P450 Enzymes. In order to further evaluate the contribution
of individual P450 forms to thio-TEPA activation in uninduced
liver microsomes, eight purified rat hepatic P450s were assayed
for thio-TEPA metabolism after reconstitution with NADPH
P450 reducÃ-ase(Table 1). Although the phenobarbital-inducible
IIB1 is at least 4-5-fold more active in thio-TEPA biotransfor
mation than any of the other P450s, P450 forms IIC11 and
IIC6 also oxygenated thio-TEPA at significant rates. In all
METABOLISM
cases TEPA was the sole detectable metabolite. Four other
P450 forms showed significantly lower activity (<0.15 nmol
TEPA formed/min/nmol
P450). Purified IIB2 also metabo
lized thio-TEPA; however, antibody inhibition experiments
described above and the low abundance of this P450 in unin
duced liver make it unlikely that this P450 contributes signifi
cantly to thio-TEPA metabolism in the uninduced microsomes.
The higher turnover exhibited by IIC11 as compared to IIC6
(Table 1) is consistent with the greater catalytic contribution of
IIC11 to microsomal thio-TEPA metabolism demonstrated by
the antibody inhibition studies. Apparent Km and Vmaxvalues
for purified P450 IIC11 were estimated to be 0.33 mM and 2.3
mol TEPA formed/min/mol P450, respectively.
Effects of in Vivo Cisplatin Treatment on Thio-TEPA Metab
olism Catalyzed by Isolated Rat Liver Microsomes. In vivo
treatment of adult male rats with cisplatin leads to a 90%
depletion of liver microsomal IIC11, whereas IIC6 levels are
moderately increased (18). Examination of thio-TEPA metab
olism catalyzed by liver microsomes prepared from cisplalintreated rats revealed that cisplatin causes a progressive suppres
sion of microsomal thio-TEPA metabolism capacity (Fig. 3/1).
By the 7th day after cisplatin administration, microsomal
TEPA formation was reduced by about 70%, i.e., to a level
similar to that of the untreated female microsomes. Cisplatin
treatment of female rats did not significantly alter microsomal
thio-TEPA metabolism (Fig. 35), in agreement with the obser
vation that IIC6, the major thio-TEPA-metabolizing
P450 in
the female liver microsomes, is not significantly affected by
cisplatin treatment in this sex (data not shown). These findings
are consistent with our conclusion, based on antibody inhibition
and purified enzyme experiments, that P450s IIC11 and IIC6
both contribute to microsomal thio-TEPA metabolism.
Inactivation of P450 IIC11 during the Course of Thio-TEPA
Metabolism. P450 UBI is susceptible to a mechanism-based
(suicidal) inactivation during the course of its oxygénationof
thio-TEPA to yield TEPA (13). Since P450 IIC11 also metab
olizes thio-TEPA to TEPA, the influence of thio-TEPA metab
olism on the activity of P450 IIC11 was examined. As shown
in Fig. 4, metabolism of thio-TEPA by purified, reconstituted
IIC11 results in a NADPH- and time-dependent inactivation
of the cytochrome. NADPH P450 reducÃ-asewas not signifi
cantly affected under these conditions of incubation (data noi
shown). When compared to UBI, the thio-TEPA-dependent
inactivation of IIC 11 was slower (Fig. 4), perhaps resulting
from the lower turnover exhibiled by IIC11 (Table 1). These
observations, togelher wilh our previous sludies on the inactiTable 1 Thio- TEPA metabolism catalyzed by purified, reconstituted rat liver
P-450 enzymes"
enzymeUBI
P-450
TEPA/min/nmol
P-4505.77
(PB-4)*
±1.74
IIB2(PB-5)
1.28 ±0.29
IIC11 (2c)
1.07 ±0.24
IIC6(PB-1)
0.30 ±0.02
IA1 (c)
0.12
IIA1 (3)
0.15
IIC7 (f)
<0.10
IIC12(2d)nmol
<0.10
" Ten pmol of each P-450 were reconstituted with 4 >igof sonicated dilauroylphosphatidylcholine and 0.7 unit of NADPH P-450 reducÃ-asefor 20 min at 20"C
immediately prior to assay. The reconstituted enzymes were incubated at 37°C
with 0.1 mivithio-TEPA and 1 mM NADPH. Aliquots were removed for TEPA
determination by gas chromatography at 5 min, except for P-450 forms IA1,
HAI. IIC7, and IIC 12, which did not show significant TEPA formation at 5 min,
and the analyses were performed at 15 min instead. Activities shown for the four
more active P-450s are mean ±SD (n = 2-6 determinations).
* Systematic P-450 designations (12) are related to trivial P-450 designations
used in earlier studies (6, 11, 13) and shown in parentheses.
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MODULATION
OF HEPATIC THIO-TEPA METABOLISM
A. MALE
3d
7d
20
40
60
80
REACTIONTIME (min)
B. FEMALE
2
0.3d,7d
20
40
60
80
100
REACTIONTIME (min)
Fig. 3. Effect of i/i vivo cisplatin administration on liver microsomal thioTEPA metabolism. Liver microsomes isolated from male (A) and female (fi) rats
sacrificed 0, 3, or 7 days after a single i.v. injection of cisplatin [0.6 mg/100 g
body weight (18)] were incubated with thio-TEPA [0.5 ITIM]and NADPH (1 mM).
Aliquots of each incubation were analyzed for TEPA formation at the indicated
time points as described under "Materials and Methods."
100
•
o
"CU
(-TT)
UBI (-TT)
80
60
O
o
alter expression of one or more of these liver P450s. Plasma
thio-TEPA levels were found to decay in a biphasic manner
following a single i.v. injection of the drug, with the decrease
more rapid in adult male rats (/•/,
~ 20 min and 70 min for a
and ßphases, respectively) than in adult female rats (/./, ~ 40
and 100 min for «and ßphases, respectively) (Fig. 5A). This
more rapid clearance of thio-TEPA in males primarily reflects
a more active metabolism of thio-TEPA to TEPA in this sex,
as evidenced by the 3-4-fold higher peak plasma levels of TEPA
in the males (Fig. 5B). This sex difference can be explained by
the absence of the thio-TEPA-metabolizing P450 IICl 1 in the
females. The data shown in Fig. 5B also indicate that higher
TEPA plasma concentration x time values are probably at
tained in the males. This suggests that in female rats, and
perhaps also in male rats under conditions of reduced or com
promised liver thio-TEPA metabolism, the time-dependent de
crease in plasma thio-TEPA levels in part reflects clearance of
the drug by pathways that do not involve its oxidation to TEPA.
These observations led us to examine whether a further
increase in plasma TEPA levels could be achieved by prior
treatment of the rats with clofibrate, a hypolipidemic drug that
significantly increases hepatic levels of an active P450 catalyst
of thio-TEPA oxidation, namely P450 UBI. Indeed, clofibrate
decreased the tv,a for thio-TEPA elimination from 20 min to
less than 4 min (Fig. 6A), while it elevated the peak plasma
levels of TEPA up to 4-fold higher than in uninduced adult
male rats (Fig. 6B). A similar increase in the rate and extent of
thio-TEPA metabolism was achieved by pretreatment of the
rats with phénobarbital(tv,a ~8 min), another effective inducer
of P450 UBI (Fig. 6). These studies demonstrate that thioTEPA metabolism and pharmacokinetics can be modulated in
the rat model using drug inducers of specific hepatic P450s.
IIC11
40
200 A. THIO-TEPA
C
O
CJ
100
A
10
Incubation
20
!
IIB1
o
—
30
Time (min)
Fig. 4. Time-dependent suicidal inactivation of purified P450 IICl 1 and P450
UBI upon incubation in the presence of thio-TEPA. Purified, reconstituted P450
proteins were preincubated at 37°Cwith NADPH and thio-TEPA (0.1 m\i) for
0 to 30 min. At the end of the preincubation, 7-ethoxycoumarin (1 mM) was
added and residual P450 activity was then determined using a fluorescent assay
for 7-ethoxycoumarin O-deethylation, as described previously ( 13). Control preincubations demonstrated that the time-dependent inactivations of these P450s did
not occur in the absence of thio-TEPA (-7T) or in the absence of NADPH (not
shown) and thus represent a mechanism-based inactivation of the P450 enzyme.
vation of the phenobarbital-inducible P450 UBI (13), suggest
that thio-TEPA may inactivate the cytochromes that participate
in the metabolism of thio-TEPA in both uninduced and phenobarbital-induced rats.
Influence of Hepatic Microsomal Monooxygenase Status on
Thio-TEPA Pharmacokinetics. In view of the differences in in
vitro thio-TEPA metabolic activity among the three P450s that
contribute to microsomal thio-TEPA oxygénation,the metab
olism of thio-TEPA in vivo was studied in uninduced rats of
both sexes and in rats that had been treated with drugs that
greatly increase hepatic levels of P450 UBI. These studies were
designed to evaluate whether thio-TEPA metabolism and pharmacokinetics in the whole rat are influenced by factors that
Õ?
10
ET
60
120
1BO 240
TIME (min)
FEMALE
60
120
180
240
TIME (min)
Fig. 5. Influence of hepatic monooxygenase status on thio-TEPA and TEPA
pharmacokinetics. Plasma levels of thio-TEPA (A) and TEPA (B) in uninduced
male and female rats were quantitated following i.v. bolus injections of thioTEPA at 2 mg/100 g body weight. Each data point represents plasma drug and
metabolite levels for a single rat sacrificed at the indicated time point. Blood
samples were analyzed as described under "Materials and Methods."
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MODULATION
OF HEPATIC THIO-TEPA
A. THIO-TEPA
i
g
CLF
0
20 40 60 80 100
TIME (min)
200
B. TEPA
CLF
O
20
40
60
80
100
TIME (min)
Fig. 6. Modulation of thio-TEPA metabolism and pharmacokinetics by the
P4SO IIBI-inducing agents clofibrate and phénobarbital.Shown are plasma levels
of thio-TEPA (A) and TEPA (A) measured in uninduced adult male rats (UT) or
in rats induced with clofibrate (CLF) or phénobarbital(PB) as described under
"Materials and Methods." Rats were given a single i.v. injection of thio-TEPA at
1 = 0 min as detailed in Fig. 5. Data points are for individual rats sacrificed at
each time point, except for the data points shown for the clofibrate-pretreated
rats sacrificed at 15. 30, and 60 min, each of which represents mean ±SD (hars)
plasma drug levels for n = S individual rats.
DISCUSSION
Earlier studies from this laboratory have established that the
oxidative biotransformation of thio-TEPA to TEPA is a cytochrome P450-dependent process and that P450 form UBI is
the principal catalyst of this desulfuration reaction in phenobarbital-induced rat liver (13). This report extends those studies
to the identification of the specific P450 enzymes that catalyze
thio-TEPA metabolism in uninduced liver tissue. In adult male
rat liver microsomes, P450 IIC11 was shown to catalyze about
60-70% and P450 IIC6 about 15-20% of the total TEPA
formation activity, while in adult female rat liver, where the
male-specific IICI 1 is absent, microsomal thio-TEPA metabo
lism proceeds at about one-third the rate of the males and IIC6
catalyzes ~70% of the total activity. These conclusions are
based on experiments with P450 form-specific inhibitory anti
bodies, on the effects of cisplatin-induced depletion of hepatic
IIC11 on thio-TEPA metabolism, and by direct demonstration
of the conversion of thio-TEPA to TEPA catalyzed by purified,
reconstituted P450s IIC11 and IIC6.
The P450 enzymes identified in this study as major catalysts
of thio-TEPA metabolism in uninduced rat liver are the same
enzymes that were previously shown to be the principal con
tributors to hepatic microsomal cyclophosphamide metabolism
(6). Moreover, P450 IIB1, which is present in phenobarbitalinduced but not uninduced rat liver, is more active than P450
IIC11 or P450 IIC6 with both drugs and hence is the major
catalyst of both thio-TEPA and cyclophosphamide metabolism
in phenobarbital-induced rats. These findings suggest a possible
structural and functional similarity at the active sites of these
METABOLISM
three P450s with respect to metabolism of these two cancer
chemotherapeutic agents. Clear differences in the activity and
specificity of these P450s with respect to steroidal substrates
are, however, apparent (11). The present observations also raise
the possibility of drug interactions between thio-TEPA and
cyclophosphamide at the level of competition for available sites
for drug activation when the two agents are administered si
multaneously, such as in some high dose therapy protocols
associated with autologous bone marrow transplantation (27,
28). The potential for an indirect drug interaction between thioTEPA and cisplatin is suggested by the present demonstration
that liver microsomes isolated from cisplatin-treated adult male
rats have much reduced capacity for P450-dependent conver
sion of thio-TEPA to TEPA. This can readily be explained by
the loss of the thio-TEPA-metabolizing P450IIC11 from livers
of rats treated with cisplatin (18). A similar interaction can be
anticipated in the case of thio-TEPA and cyclophosphamide,
insofar as the latter compound can also suppress hepatic P450
IIC11 to a significant extent [~75% decrease (29)].
Studies performed with purified, reconstituted P450 IIC11
as well as microsomal assays (data not shown) demonstrated
that this cytochrome is susceptible to suicide inactivation by
thio-TEPA in a manner similar to that of P450 UBI (13). ThioTEPA-dependent inactivation of IIC11 proceeded more slowly
than that of UBI. This slower inactivation of IIC11 may reflect
the 15-fold higher apparent Km [0.33 HIM) and 5-fold lower
apparent Vmax(2.3 mol TEPA formed/min/mol IIC11) exhib
ited by IIC11 as compared to UBI (Km (app) = 0.02 ITIMand
Vmax(app) = 11 mol thio-TEPA metabolized/min/mol
P450
(13)].
In order to ascertain whether the observed sex differences
and P450 induction effects on in vitro thio-TEPA metabolism
impact on drug pharmacokinetics (and conceivably drug action
and toxicity as well), plasma levels of thio-TEPA and TEPA
were monitored in uninduced adult male and female rats, as
well as in male rats pretreated with phénobarbital or with
clofibrate, a hypolipidemic agent that significantly elevates
hepatic levels of the thio-TEPA-metabolizing P450 UBI (6). In
agreement with predictions based on the in vitro microsomal
studies, marked sex differences in thio-TEPA clearance were
found, with male rats being more efficient (tv,a = 20 min) than
female rats (tVla 40 min) in this process. These studies also
established that the P450 inducing agents clofibrate and phé
nobarbital can both accelerate thio-TEPA clearance (lv,a = 48 min). In all cases, increased plasma thio-TEPA clearance was
accompanied by a corresponding increase in plasma TEPA
levels, and probably also an increase in the plasma area under
the curve values for this oxo-metabolite. These pharmacoki
netics are in accord with the relative thio-TEPA metabolism
rates measured in vitro with isolated liver microsomes and
provide good evidence that hepatic metabolism by these P450
enzymes is a key determinant of thio-TEPA biotransformation
and clearance in vivo. The pharmacokinetic data obtained in
this study also indicate that thio-TEPA metabolism/elimina
tion is complex, perhaps biphasic, in rats, as it is in cancer
patients (17). This may reflect, in part, suicide inactivation in
vivo of the P450 enzymes active in thio-TEPA metabolism.
While thio-TEPA and TEPA both possess significant antitumor activity (30, 31), we have previously reported that the
two compounds act by somewhat different mechanisms (14)
and conceivably may have differing toxicities. In addition,
P450-dependent metabolism of thio-TEPA yields other reactive
metabolites that contribute to microsomal enzyme inactivation
2344
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MODULATION
OF HEPATIC THIO-TEPA
(13) and may also enhance drug cytotoxicity toward cultured
tumor cells (14). TEPA itself, however, does not appear re
sponsible for these latter two effects of thio-TEPA. Although
the consequences of thio-TEPA metabolism by hepatic P450
enzymes are still not completely understood, changes in the
rate and extent of thio-TEPA metabolism seem likely to impact
on the antitumor activity of this chemotherapeutic drug. The
present characterization of P450-inducing agents that serve as
effective modulators of thio-TEPA metabolism and pharmacokinetics should provide the opportunity to test this hypothesis
and to explore approaches to modulate the efficacy of thioTEPA in patients treated with this cancer chemotherapeutic
agent.
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N,N′,N″-Triethylenethiophosphoramide (Thio-TEPA)
Oxygenation by Constitutive Hepatic P450 Enzymes and
Modulation of Drug Metabolism and Clearance in Vivo by
P450-inducing Agents
Sze-fong Ng and David J. Waxman
Cancer Res 1991;51:2340-2345.
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