![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 287, Issue 2, 791-799, November 1998
Department of Drug Metabolism and Molecular Toxicology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji-shi, Tokyo 192-03, Japan
 |
Abstract |
|---|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
A toxicokinetic study was performed using rats to investigate the possible mechanism of 18 acute deaths in Japanese patients with cancer and herpes zoster by interactions of the new oral antiviral drug, sorivudine (SRV), with one of the oral 5-fluorouracil (5-FU) prodrugs within 40 days after approval of the use of SRV. Tegafur, an anticancer 5-FU prodrug suggested to be used by most of the patients who died, and SRV were orally administered to rats simultaneously once daily. All of these rats died within 10 days, whereas rats given SRV or tegafur alone under the same dosage conditions showed no appreciable change over 20 days compared with controls. In the rats given both drugs, bone marrow and intestinal membrane mucosa were greatly damaged at an early stage of the coadministration, and before death, the animals showed marked decreases in white blood cell and platelet counts, diarrhea with bloody flux, and severe anorexia, as was also manifested by the patients who subsequently died. In the rats given both drugs for 6 days, extremely enhanced 5-FU levels were observed from the first day of administration in plasma and in all tissues examined, including bone marrow and intestines. The extreme enhancement of the tissue 5-FU levels was attributable to the facile inactivation by (E)-5-(2-bromovinyl)uracil (BVU) of hepatic dihydropyrimidine dehydrogenase (DPD), a key enzyme regulating the systemic 5-FU level in the rat and human. BVU, a major metabolite formed from SRV by gut flora, was found at considerable levels in the liver of rats orally administered SRV alone or SRV and tegafur, and there was a marked decrease in hepatic DPD activity. In the presence of NADPH, DPD purified from rat liver cytosol was rapidly and irreversibly inactivated by [14C]BVU as a suicide inhibitor with concomitant incorporation of the radioactivity into the enzyme protein, although SRV showed no inhibitory effect on DPD under the same conditions. Human liver DPD was recently demonstrated by us to be inactivated with BVU in a manner very similar to rat DPD.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
The
Pharmaceutical Affairs Bureau, Japanese Ministry of Health and Welfare,
reported that in 1993 fifteen deaths in Japanese patients occurred in
association with the coadministration of the new antiviral drug for
herpes zoster, SRV, with one of oral 5-FU prodrugs within 40 days after
SRV was approved by the Japanese government and began to be used
clinically (Pharmaceutical Affairs Bureau, 1994
). In addition, the
report noted that three additional patients died from the same drug
interactions during phase II clinical trials of SRV. The report also
mentioned that before death, all of these patients had severe symptoms
of toxicity, such as diarrhea with bloody flux and marked decreases in
white blood cell and platelet counts and that eight other Japanese
patients receiving both drugs during this period had severe symptoms of gastrointestinal- and myelo-toxicity. All of these patients had received SRV for a period of several days although being administered continuous anticancer chemotherapy with one of the 5-FU prodrugs.
The clinical dose of SRV, 50 mg three times a day, is much lower than
the minimal toxic doses for animals, e.g., >2000 mg/kg in
rats (Nagasaka et al., 1990). Therefore, there were no
reports of acute death or toxic symptoms as described above in patients who had herpes zoster and had received SRV alone or SRV and anticancer drugs other than 5-FU or its oral prodrugs, including FT. FT, the most
widely used oral 5-FU prodrug in combination with uracil in Japan, is
activated to 5-FU mainly by hepatic cytochrome P-450 after being
absorbed from the intestinal membrane (Fujita et al., 1976).
Actually, most of the patients who died from the drug interaction were
likely to receive FT, although a few seemed to receive one of the other
types of 5-FU prodrugs, such as carmofur
(1-hexylcarbamoyl-5-fluorouracil) and doxifluridine
(5'-deoxy-5-fluorouridine).
The anticancer drug, 5-FU, and its oral prodrugs have been well recognized to have severe toxic effects on the gastrointestinal tract and bone marrow, both of which have rapid cell proliferation, in patients on long-term treatment at clinical dosage levels. These toxic effects on the human and animals are characteristic of 5-FU as an inhibitor of DNA synthesis.
The 18 patient deaths would not have occurred if the following three
facts had been carefully considered in the safety/risk assessment of
drug interactions during development of the new antiviral drug SRV.
First, BVU is a major metabolite of SRV in rats (Nishimoto et
al., 1990) and humans (Ogiwara et al., 1990). Second,
BVU markedly increased the plasma concentration of 5-FU and enhanced
the toxicity of 5-FU with concomitant retardation of hepatic DPD
activity when 5-FU and BVU were given only once successively i.p. to
rats and mice (Desgranges et al., 1986). Third, an in
vitro study using DPD partially purified from rat liver
demonstrated that the enzyme was inactivated with BVU in the presence
of NADPH and that its activity was not restored by dialysis (Desgranges
et al., 1986), although the inactivation mechanism is equivocal.
Hepatic DPD, a homodimeric 210-kDa flavoprotein with Fe-S clusters, has
been recognized as a key enzyme regulating plasma and tissue
concentrations of 5-FU administered to humans (Lu et al.,
1992) as well as to rats (Shiotani and Weber, 1981; Fujimoto et
al., 1991; Lu et al., 1993a). 5-FU is dihydrogenated at
the 5,6-double bond by DPD and is rapidly decomposed to
-fluoro-
-alanine by subsequent enzymatic processes (Diasio and
Harris, 1989). In the human, more than 85% of 5-FU administered i.v.
is catabolized by DPD through this metabolic pathway (Diasio and
Harris, 1989). The aforementioned facts imply that the 18 patient
deaths might be due to an increase in the tissue 5-FU level by
inactivation of hepatic DPD with BVU formed from the antiviral SRV that
was coadministered.
Recently, we have undertaken a study on the possible mechanism of
the patient deaths using rats and briefly reported in a communication
that BVU rapidly inactivated DPD in the presence of NADPH by covalent
binding of a reduced form as a reactive metabolite of BVU to the enzyme
protein (Okuda et al., 1997). We also reported that oral
coadministration of SRV and FT to rats elevated their blood and tissue
levels of 5-FU, resulting in severe damage to bone marrow and
intestinal membrane. More recently, we demonstrated that human DPD was
also inactivated by BVU in the same manner as was rat hepatic DPD
(Ogura et al., 1998).
The report deals with 1) detailed results of our toxicokinetic study of 5-FU in the bone marrow and small intestine as well as in the plasma and liver of rats orally administered FT and SRV, 2) histological changes in these tissues of rats orally administered both drugs in addition to the hematological and toxicological findings in the animals and 3) the mechanism of the irreversible inactivation of purified rat liver DPD using [14C]BVU.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Materials.
SRV was prepared as reported previously (Sakata
et al., 1980). [14C]BVU was prepared from
5-formyluracil with [2-14C]malonic acid (9.25 MBq, Du
Pont NEN Research Products, Boston, MA) via
5-(2-carboxy-[2-14C]vinyl)uracil in the same manner as
used for the synthesis of unlabeled BVU (Jones et al.,
1979). [14C]BVU synthesized had a specific activity of
2.0 MBq/µmol and a radiochemical purity of more than 99% after
purification by HPLC.
Animal treatment. Female Wistar rats (5 groups of 90 animals each, 6 wk of age) were obtained from Japan SLC, Inc. (Hamamatsu, Japan) and housed with free access to food and water in a light- and dark-controlled room (lights on 6:00 A.M. to 6:00 P.M. and lights off 6:00 P.M. to 6:00 A.M.). The animals were orally administered FT alone (60 mg/kg, triple the daily dose in clinical use) (Taiho Pharmaceutical Co., Tokushima, Japan), SRV alone (30 mg/kg, 12 times the daily dose in clinical use), BVU alone (3.7 mg/kg) (Sigma Chemical Co., St. Louis, MO) or FT (60 mg/kg) and SRV (30 mg/kg) simultaneously at 9:00 to 10:00 A.M. once daily for 6 days as a 1-ml suspension in 0.5% (w/v) sodium carboxymethylcellulose. The same volume of the vehicle was also used for the control animals. Blood and tissue samples were collected at 1, 2, 4, 8 and 24 hr after administration of the drugs on days 1, 2, 4 and 6. These samples were collected from three animals at each time point after they were anaesthetized with ether and killed.
Quantification of pyrimidine levels.
FT, 5-FU and uracil in
the plasma, urine and tissue samples were determined as reported
previously (Marunaka et al., 1980). SRV and BVU were
analyzed by HPLC on an Inertsil ODS-2 column (150 × 4.6 mm, GL
Sciences, Tokyo, Japan) eluted at a flow rate of 1 ml/min with 15%
(v/v) acetonitrile-water containing 0.01% (w/v) trifluoroacetic acid
after extraction with ethyl acetate from plasma (1 ml) and liver
homogenates (0.3 g tissue/1 ml saline). SRV and BVU eluted at retention
times of 7.4 and 9 min, respectively, from the HPLC column were
determined with a detection limit of 0.1 µg/ml plasma or g liver as
reported previously (Okuda et al., 1997).
Tissue preparation for optical microscopy. Intestines and other tissues were obtained at the 24th hr on days 3 and 6 from the rats after the repeated oral administration of the vehicle, FT alone, SRV alone or FT and SRV at the same dose(s) as mentioned above. The tissues were fixed in a solution of 10% (w/v) formalin, dehydrated, and embedded in paraffin, and paraffin sections (5 µm in thickness) were stained with eosin and hematoxylin in the usual manner.
CFU-GM assay.
Bone marrow cells were collected at the 24th
hr on days 1, 2, 4 and 6 from femurs of rats after repeated oral
administration of the vehicle, FT alone, SRV alone or FT and SRV at the
same dose(s) as mentioned above, seeded at 105 mononuclear
cells/35-mm Petri dish, and cultured in the presence of a recombinant
mouse granulocyte-macrophage colony-stimulating factor under the same
conditions as reported previously (Okuda et al., 1997).
After incubation for 7 days at 37°C in a humidified atmosphere of 5%
(v/v) CO2, colonies containing 40 cells were counted as
CFU-GM colonies with an inverted microscope.
Enzyme assay.
DPD was purified from young adult female
Wistar rats as reported previously (Lu et al., 1993a).
Activity of the purified enzyme toward 5-FU was assayed by a previously
reported method (Desgranges et al., 1986) with
modifications; the enzyme (5-50 ng of protein/5 µl) was incubated
with 20 µM [14C]5-FU (2.1 MBq/µmol, Moravek
Biochemicals Inc., Brea, CA) at 37°C for 5 min in the presence of 200 µM NADPH, 2.5 mM magnesium chloride and 10 mM 2-mercaptoethanol in a
final volume of 50 µl of 30% (v/v) glycerol-35 mM K-phosphate
buffer, pH 7.4. [14C]H2-5-FU formed as a
metabolite was separated on a diethylaminoethyl cellulose TLC plate as
reported previously (Traut and Loechel, 1984).
) with modifications; rat liver
cytosol was incubated with 25 µM [14C]5-FU (29.6 kBq/mmol) in the presence of 500 µM NADPH, 1.25 mM magnesium chloride
and 5 mM 2-mercaptoethanol in a final volume of 250 µl of 35 mM
K-phosphate buffer, pH 7.4, at 37°C for 10 min. After the termination
of the enzyme reaction by the addition of 60 µl of 2 M aqueous
potassium hydroxide, the [14C]H2-5-FU formed
was hydrolyzed to [14C]-fluoro--ureidopropionate
and/or [14C]-fluoro--alanine as reported previously
(Ikenaka et al., 1979). [14C]-Fluoro--ureidopropionate and
[14C]-fluoro--alanine were separated from the
substrate, [14C]5-FU, on a silica TLC plate, and their
radioactivities were determined by liquid scintillation counting.
Reaction of purified DPD with BVU. DPD (0.5 µg) purified from rat liver cytosol was preincubated with various concentrations of BVU in the presence and absence of 200 µM NADPH in a final volume of 50 µl of 30% (v/v) glycerol-35 mM K-phosphate buffer, pH 7.4, containing 2.5 mM magnesium chloride and 2.5 mM 2-mercaptoethanol at 37°C for 5 min. Aliquots (5 µl) of the reaction mixture were withdrawn and immediately assayed for residual DPD activity. A 10% decrease in the remaining activity was observed with the purified DPD preincubated with 50 µM BVU in the absence of NADPH when the preincubation mixture was diluted 10-fold and incubated for the enzyme assay with [14C]5-FU in the presence of NADPH (200 µM). The apparent decrease at various concentrations of BVU was subtracted from the data on the remaining activity to show that DPD was not influenced by BVU in the absence of NADPH during the preincubation.
For isolation of the DPD protein incorporating the radioactivity of 4 µM [14C]BVU, the final volume of the incubation mixture was increased up to 500 µl without changing the concentrations of the constituents. The mixture was incubated at 37°C for 30 min, diluted with a large excess (25 times) of BVU and then rapidly chilled in an ice bath. The radioactivity incorporated into the enzyme protein was separated from unreacted [14C]BVU by HPLC and determined by liquid scintillation counting.HPLC of DPD. HPLC was carried out on a TSK GEL 2000 SWXL column (300 × 7.8 mm) (Tosoh Corporation, Tokyo, Japan) with a Gilson model 325 HPLC pump (Villiers-le-Bel, France). Rat liver DPD was eluted at a flow rate of 1 ml/min with 35 mM K-phosphate buffer, pH 7.4, at a retention time of 5.2 min. The DPD isolated by HPLC after incubation with 4 µM [14C]BVU for 30 min in the presence of NADPH was concentrated with a Centricon concentrator (Amicon, Inc., Beverly, MA) and rechromatographed under the same chromatographic conditions. The chromatogram was monitored by absorbance at 220 nm with a Gilson model 111B UV detector and by liquid scintillation counting of the column effluent collected every 30 sec.
Electrophoresis.
SDS-PAGE was carried out on a 10% (w/v)
polyacrylamide gel slab by the previously reported method
(Laemmli, 1970). Native PAGE was carried out on a 9% (w/v)
polyacrylamide gel slab as reported previously (Lu et al.,
1992). Proteins were stained with Coomassie Brilliant Blue R250.
Radioluminography. A radiolabeled DPD fraction eluted from the HPLC column was subjected to SDS-PAGE after concentration of the effluent with a Centricon concentrator. After staining as mentioned above, the gel slab was dried in vacuo. Radioactivity migrating with DPD on the gel slab was visualized by radioluminography, using a Fuji Photo Co. model BAS 2000 (Tokyo, Japan) after exposing the dried gel slab to a Fuji imaging plate for 8 hr.
Statistical analysis. The significance of differences was determined by Student's t test.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Marked increases in plasma and tissue levels of 5-FU in rats coadministered FT and SRV. Toxicokinetics were investigated for the active metabolite 5-FU formed from its prodrug FT in rats orally coadministered FT (60 mg/kg) and SRV (30 mg/kg) simultaneously once daily for 6 days. In addition, plasma and urinary levels of uracil, an endogenous substrate for DPD, were also determined in the animals.
The daily doses of FT and SRV coadministered to the rats were determined through several preliminary experiments by adjusting the days at which severe toxicity appeared and death occurred in the rats to those reported for the patients: about 3 to 4 days after coadministration for toxicity and by 10 days for death. At these doses, a 6-day coadministration had to be selected for our toxicokinetic study because one third of the animals died on days 6 to 7 of treatment, and an increasing number of deaths occurred thereafter with prolongation of the duration of treatment. FT was absorbed rapidly, with a Tmax of 1 to 2 hr, in rats orally administered FT alone or FT and SRV once daily and almost disappeared from plasma, with a t1/2 of 2.7 to 4.3 hr, within 24 hr throughout the days examined (fig. 1A). The plasma FT level in the rats coadministered FT and SRV showed no appreciable difference from that in the animals administered FT alone. Plasma AUC0-24 and Cmax of FT in the rats administered both drugs showed the almost same magnitudes as those in animals administered FT alone.
|
|
|
Marked decrease in hepatic DPD activity in rats coadministered FT and SRV. There was no appreciable difference in hepatic DPD activity (1.0-1.2 nmol 5-FU reduced/mg protein/min) between control rats and rats administered FT alone once daily. However, the DPD activity in rats orally administered FT and SRV once daily for 6 days was markedly decreased to 34.2, 21.1 and 8.9% that of the controls 24 hr after coadministration on days 1, 4 and 6, respectively.
SRV and its metabolite BVU were detected at significant levels in the plasma and liver of rats orally given SRV alone as well as FT and SRV. For SRV, plasma and hepatic Cmax occurred 2 hr after dosage and averaged 23.3 nmol/ml (range: 18.3-30.6 nmol/ml) and 34.3 nmol/g tissue (range: 21.5-59.5 nmol/g tissue), respectively, throughout the days examined. For BVU, plasma and hepatic Cmax were reached at 8 hr after dosage of SRV and averaged 12.6 nmol/ml (range: 10.8-14.7 nmol/ml) and 25.5 nmol/g tissue (range: 22.3-31.4 nmol/g tissue), respectively. SRV and BVU were cleared from the plasma and liver with t1/2 of 1.5 to 5 and 9 to 14 hr, respectively, throughout the days examined. In rats orally administered BVU at a dose of 3.7 mg/kg once daily for 6 days, Cmax of BVU in the plasma and liver were 13.2 nmol/ml and 27.0 nmol/g tissue, respectively, which were approximately equal to those from SRV orally administered once daily at a dose of 30 mg/kg. In the animals administered BVU at the aforementioned dose, hepatic DPD activity was markedly decreased throughout the days examined, e.g., the DPD activity was 14% of the controls on day 6.Marked increase in toxicity by increased tissue levels of 5-FU in rats coadministered FT and SRV. Body weight and dietary intake of rats orally administered FT and SRV once daily for 6 days decreased after day 2 (fig. 3). From the third day, body weight rapidly deceased, accompanied by a marked decrease in dietary intake. On days 5 to 6, the dietary intake was negligible, and on days 6 to 7, one third of the rats administered FT and SRV died; none of the animals remained alive on day 10. However, the animals given the same dose of FT or SRV alone once daily for 20 days showed no appreciable change in vital signs compared with the control animals.
|
|
|
|
Inactivation of DPD by covalent binding of BVU in the presence of NADPH. DPD, isolated from rat liver cytosol and purified to homogeneity, had an activity to catalyze the hydrogenation of [14C]5-FU to [14C]H2-5-FU at a rate of 816 nmol/mg protein/min in the presence of NADPH. Native PAGE of the purified enzyme protein showed a single band with an apparent molecular mass of 210 kDa, whereas the enzyme migrated as two bands with molecular masses of 105 and 97.7 kDa on SDS-PAGE.
The DPD activity was strongly inhibited by preincubation with BVU in the presence of NADPH for 5 min before incubation with the substrate [14C]5-FU for determining the enzyme activity (fig. 6). Under the conditions used, IC50 of BVU for inactivation of DPD was 4 µM. Omitting the cofactor NADPH from the preincubation medium, BVU showed no inhibitory effect on DPD. However, in the presence of NADPH, SRV showed no inhibitory effect on the 5-FU-reducing activity of the DPD even at 50 µM under the same preincubation conditions (fig. 6).
|
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Our study using rats strongly suggests that the 18 patient deaths caused by the interactions of the 5-FU prodrugs with the new antiviral drug SRV were due to extremely high concentrations of 5-FU in various tissues, especially in bone marrow and intestines, as a result of the facile inactivation of hepatic DPD by the covalent binding of an active metabolite formed in liver from BVU which is generated from SRV (fig. 8).
|
Female Wistar rats were required to be used in our study to
reinvestigate an incomplete study reported by previous workers who were
engaged in the SRV development. After the 18 patient deaths, they
(Yoshifune et al., 1994) reported, without showing any data
on the 5-FU levels in plasma and tissues, that neither diarrhea with
bloody flux nor deaths occurred in female Wistar rats by repeated oral
coadministration of FT and SRV under the same dosage conditions,
including the same volume of the same vehicle for suspending both
drugs, as used in our study. However, our study, including several
preliminary experiments for determination of the dosage conditions,
demonstrated that all the animals given both drugs died following the
severe diarrhea in 10 days so far as treated under the same dosage
conditions as reported by them. They, however, also demonstrated the
marked decreases in platelet and white blood cell counts in the rats
given both drugs to the almost same extent as demonstrated in our study.
In the rat, BVU was reported to be generated from SRV by gut flora but
not to be formed in tissues and was demonstrated to show
Cmax at 8 hr in plasma after oral administration (Nishimoto et al., 1990). Similar evidence also has been provided for
the microbial formation and plasma level of BVU from SRV in the human (Ogiwara et al., 1990). Bacteroides species, such
as Bacteroides eggerthill and Bacteroides
vulgatus, which abundantly exist in human intestines, were
identified as the major bacteria which generate BVU from SRV (Nakayama
et al., 1997).
Recently, Yan et al. (1997) reported the strong suppression
of DPD activity in humans administered SRV; i.e., patients
having herpes zoster and administered SRV at a dosage of 40 mg/day for 10 consecutive days did not show any DPD activity in their peripheral blood mononuclear cells during the period of administration. During the
period of SRV administration, BVU appeared in blood at a remarkably high level and was eliminated from the circulation within 7 days after
the last dose of SRV.
DPD is most abundant in the liver and occurs at very low concentrations
in other tissues, including the colon in the human (Ho et
al., 1986) and bone marrow in the rat (Ikenaka et al., 1979). The primary structure of DPD has been elucidated by molecular cloning of cDNA encoding the porcine (Yokota et al., 1994),
human (Yokota et al., 1994), bovine (Albin et
al., 1996) and rat (Kimura et al., 1996) enzyme
subunits, which share very strong identity, more than 89%, in amino
acid sequence. The DPDs have been demonstrated to have a potential
pyrimidine-binding domain including the sole cysteinyl residue, which
is highly conserved in amino acid sequence across mammalian species
(Yokota et al., 1994).
Previously, we reported, using purified rat liver DPD and radiolabeled
BVU, that rapid inactivation of DPD by BVU occurred in the presence of
NADPH with concomitant incorporation of the radioactivity of BVU into
the enzyme protein (Okuda et al., 1997). The radioactivity
was incorporated into the DPD protein in a manner reciprocal to the
enzyme inactivation and was inseparable from the enzyme protein by HPLC
on a gel filtration column (Okuda et al., 1997). These
results indicated that the enzyme inactivation is caused by covalent
binding to DPD of a reduced form of BVU.
DPD purified from rat liver in our study showed a single band on native
PAGE but migrated as two sharp bands, an intact subunit as a major band
at 105 kDa and a degradation product as a minor one at 97.7 kDa, on
SDS-PAGE, as had already been shown with purified specimens of human
(Lu et al., 1992) and porcine (Lu et al., 1993a) DPDs; the purified enzymes were also demonstrated to be degraded, in
part, to peptides with a little smaller molecular mass under the
denaturing conditions used for SDS-PAGE. Radioactivity incorporated into DPD protein after the incubation with [14C]BVU in
the presence of NADPH was also detected in the two bands on SDS-PAGE,
after isolation on a gel filtration column. In our study, the
radioactivity was found to be inseparable from the enzyme protein even
by SDS-PAGE, after gel chromatography.
The reactive metabolite formed from BVU by DPD was suggested to act as a suicide inhibitor on the enzyme, because various thiol compounds (20 mM), such as cysteine, glutathione and dithiothreitol, added to the incubation mixture had no retarding effect on enzyme inactivation and on the radiolabeling of DPD by [14C]BVU (data not shown). It is reasonable to suppose that the reactive metabolite, dihydro-BVU (H2-BVU), would be 5-(2-bromoethyliden)uracil, a reactive allyl bromide type of electrophile, rather than the direct hydrogenation product, 5-(2-bromovinyl)-5,6-dihydrouracil, possibly with considerable stability (fig. 8). The reactive H2-BVU is formed at the potential pyrimidine-binding domain of DPD, and it might react instantly with the sulfhydryl group of the sole cysteinyl residue in the domain.
The above hypothesis on the suicide inhibition of DPD may be supported
by the fact that 5-iodouracil (Porter et al., 1991) and
5-ethynyluracil (Porter et al., 1992) inactivate bovine
liver DPD in the presence of NADPH by covalent binding of their
reactive metabolites to the sulfhydryl group of the cysteinyl residue
located in the pyrimidine-binding domain of the enzyme.
Very recently, we demonstrated, using recombinant human liver DPD
expressed in Escherichia coli and purified to homogeneity, that BVU covalently binds to and inactivates the DPD in the presence of
NADPH (Ogura et al., 1998). Radioactivity of
[14C]BVU was incorporated in the presence of NADPH into
the human DPD with concomitant loss of enzyme activity. In the absence
of NADPH, the human DPD was not inactivated nor radiolabeled. SRV showed no inhibitory effect on DPD in the presence of NADPH as demonstrated with rat DPD in the present study.
5-FU is as good a substrate for DPD as are the endogenous pyrimidines,
uracil and thymine (Lu et al., 1993a). Tissue 5-FU levels in
patients administered anticancer chemotherapy with 5-FU or its prodrugs
are strongly suggested to depend on hepatic DPD activity (Diasio and
Harris, 1989). Actually, patients with very low genetically determined
hepatic DPD activity have been reported to die during anticancer
chemotherapy by 5-FU infusion (Lu et al., 1993b; Milano and
Etienne, 1994). Therefore, it has been noted that 5-FU-based anticancer
chemotherapy should not be administered to patients who are
DPD-deficient or have very low DPD activity in liver or in peripheral
blood mononuclear cells, for otherwise they will suffer from severe
toxic symptoms or die from markedly elevated tissue 5-FU levels (Lu
et al., 1993b; Chazal et al., 1996).
The clinically observed typical toxic symptoms of 5-FU and its
prodrugs, marked decreases in white blood cells and platelets, were
reported for all of the patients who had received both SRV and one of
the oral 5-FU prodrugs and who subsequently died (Pharmaceutical Affairs Bureau, 1994). The severe hematotoxicity mentioned above was
also found in the rats administered FT and SRV, but not in the animals
treated with the same dose of FT or SRV alone. On the basis of the
present CFU-GM assay for bone marrow in the rats given both drugs,
proliferation of bone marrow cells was suppressed at an early stage of
the coadministration.
The average Cmax of hepatic BVU generated from SRV in rats
was much higher than for the facile and almost complete inactivation of
the purified rat liver DPD in the presence of NADPH. However, liver
cytosol from the rats orally administered SRV and FT still had DPD
activity at a level of about 9% that of the controls. Moreover, in the
liver of rats orally given BVU for 6 days, the DPD activity remained at
a level of 14% of the controls on day 6. There may be two reasons for
the remaining DPD activity in the liver of the rats given FT and SRV or
BVU alone. One is the markedly elevated levels of the pyrimidines, 5-FU
and uracil as demonstrated in our study, which act as competitive
inhibitors for the inactivation of DPD by BVU. In connection with this,
the inhibition of the DPD activity by BVU in rat liver cytosol
containing NADPH has been demonstrated to be significantly retarded in
the presence of 5-FU or uracil (Desgranges et al., 1986;
Tatsumi et al., 1987). Another reason is the circadian
rhythm of hepatic DPD activity in the rat. DPD activity in rat liver
was demonstrated to vary over a 24-hr period in association with
a light-dark cycle (Harris et al., 1988, 1989). Although the
physiological mechanism for the circadian rhythm is unclear, facile
synthesis and decomposition of the DPD protein could occur.
Thus, our study provides the first evidence for lethal interaction of respective metabolites from two drugs. One of the two drugs gives an active metabolite with high toxicity, the elevated tissue levels of which readily result in death, and the other gives a metabolite that irreversibly inactivates the enzyme catabolizing the active metabolite. In other words, in case of the interaction of SRV with one of 5-FU prodrugs, the patients who died are strongly suggested to become extremely poor metabolizers for the active metabolite, 5-FU, with high toxicity as a result of irreversible inactivation of the key enzyme, DPD, by BVU from SRV. Except for the metabolic pathway depending on DPD, there is no major alternative pathway for the 5-FU catabolism in the human as well as in the rat. Studies on the synthesis of the reactive metabolite from BVU and on the mode of its covalent binding to DPD are now in progress in our laboratory.
| |
Acknowledgments |
|---|
The authors appreciate Sekio Nagayama, Kazumasa Ikeda, Shuji Yamaguchi, Takahito Nishiyama, Yoshimasa Nakamura and Yasuro Kawaguchi of Tokushima Research Center, Taiho Pharmaceutical Co., Ltd. for their great contributions to our toxicokinetic experiments.
| |
Footnotes |
|---|
Accepted for publication June 3, 1998.
Received for publication November 14, 1997.
Send reprint requests to: Dr. Tadashi Watabe, Department of Drug Metabolism and Molecular Toxicology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji-shi, Tokyo 192-03, Japan.
| |
Abbreviations |
|---|
AUC0-24, area under the curve 0 to
24 hr after administration;
BVU, (E)-5-(2-bromovinyl)uracil;
CFU-GM, colony-forming unit granulocyte-macrophage;
Cmax, maximum concentration;
DPD, dihydropyrimidine dehydrogenase;
FT, tegafur [1-(2-tetrahydrofuryl)-5-fluorouracil];
5-FU, 5-fluorouracil;
H2-BVU, dihydro-BVU;
H2-5-FU, 5,6-dihydro-5-FU;
HPLC, high-pressure liquid chromatography;
IC50, 50%
inhibitory concentration;
NADPH, nicotinamide adenine dinucleotide
phosphate, reduced form;
SDS-PAGE, sodium dodecyl
sulfate-polyacrylamide gel electrophoresis;
SRV, sorivudine
[1--D-arabinofuranosyl-(E)-5-(2-bromovinyl)uracil];
TLC, thin-layer chromatography;
Tmax, time to maximum
concentration.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
This article has been cited by other articles:
|
|
 |
|
  F. G. A. Jansman, A. J. A. Jansen, J. L. L. Coenen, J. C. de Graaf, W. M. Smit, D. T. Sleijfer, and J. R. B. Brouwers Assessing the clinical significance of drug interactions with fluorouracil in patients with colorectal cancer Am. J. Health Syst. Pharm., September 1, 2005; 62(17): 1788 - 1793. [Full Text] [PDF]
|
|
|
|
 |
|
 K. Ogura, T. Ohnuma, Y. Minamide, A. Mizuno, T. Nishiyama, S. Nagashima, M. Kanamaru, A. Hiratsuka, T. Watabe, and T. Uematsu Dihydropyrimidine Dehydrogenase Activity in 150 Healthy Japanese Volunteers and Identification of Novel Mutations Clin. Cancer Res., July 15, 2005; 11(14): 5104 - 5111. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Inada, Y. Hirao, T. Koga, M. Itose, J.-i. Kunizaki, T. Shimizu, and H. Sato RELATIONSHIPS AMONG PLASMA [2-13C]URACIL CONCENTRATIONS, BREATH 13CO2 EXPIRATION, AND DIHYDROPYRIMIDINE DEHYDROGENASE (DPD) ACTIVITY IN THE LIVER IN NORMAL AND DPD-DEFICIENT DOGS Drug Metab. Dispos., March 1, 2005; 33(3): 381 - 387. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Malet-Martino and R. Martino Clinical Studies of Three Oral Prodrugs of 5-Fluorouracil (Capecitabine, UFT, S-1): A Review Oncologist, August 1, 2002; 7(4): 288 - 323. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Balzarini and C. McGuigan Bicyclic pyrimidine nucleoside analogues (BCNAs) as highly selective and potent inhibitors of varicella-zoster virus replication J. Antimicrob. Chemother., July 1, 2002; 50(1): 5 - 9. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Balzarini, R. Sienaert, S. Liekens, A. Van Kuilenburg, A. Carangio, R. Esnouf, E. De Clercq, and C. McGuigan Lack of Susceptibility of Bicyclic Nucleoside Analogs, Highly Potent Inhibitors of Varicella-Zoster Virus, to the Catabolic Action of Thymidine Phosphorylase and Dihydropyrimidine Dehydrogenase Mol. Pharmacol., May 1, 2002; 61(5): 1140 - 1145. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Keith, X.-D. Guo, S. Zentko, N. Harold, B. Schuler, M. Quinn, J. Shapiro, and J. L. Grem Impact of Two Weekly Schedules of Oral Eniluracil Given with Fluorouracil and Leucovorin on the Duration of Dihydropyrimidine Dehydrogenase Inhibition Clin. Cancer Res., May 1, 2002; 8(5): 1045 - 1050. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Nishiyama, K. Ogura, H. Okuda, K. Suda, A. Kato, and T. Watabe Mechanism-Based Inactivation of Human Dihydropyrimidine Dehydrogenase by (E)-5-(2-Bromovinyl)uracil in the Presence of NADPH Mol. Pharmacol., May 1, 2000; 57(5): 899 - 905. [Abstract] [Full Text]
|
|
|
|
|
|
S.-i. Kanamitsu, K. Ito, H. Okuda, K. Ogura, T. Watabe, K. Muro, and Y. Sugiyama Prediction of In Vivo Drug-Drug Interactions Based on Mechanism-based Inhibition from In Vitro Data: Inhibition of 5-Fluorouracil Metabolism by (E)-5-(2-Bromovinyl)uracil Drug Metab. Dispos., April 1, 2000; 28(4): 467 - 474. [Abstract] [Full Text]
|
|
|
|
|
|
S. Sharma and L. B. Saltz Oral Chemotherapeutic Agents for Colorectal Cancer Oncologist, April 1, 2000; 5(2): 99 - 107. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
) of NADPH as described in "Materials and Methods." After
incubation, the hepatic DPD was isolated from the mixture by HPLC on a
gel filtration column as described in "Materials and Methods." A
and B represent SDS-PAGE and radioluminography, respectively, of the
radiolabeled DPD. Lane M represents molecular mass markers.
Radioactivity incorporated into the DPD was visualized by
radioluminography of the SDS-PAGE gel plate stained with Coomassie
Brilliant Blue.
