Dihydropyrimidine dehydrogenase (dihydrouracil
dehydrogenase, dihydrothymine dehydrogenase, uracil reductase, EC
188.8.131.52, DPD) is the initial rate-limiting enzyme in pyrimidine
catabolism. DPD is important in the catabolism of not only the
naturally occurring pyrimidines uracil and thymine, but also the
widely used antimetabolite cancer chemotherapy agent 5-fluorouracil
(5-FU).[1,2] DPD thus occupies an important position in the overall
metabolism of 5-FU, converting over 85% of clinically administered
5-FU to 5-FUH2, an inactive metabolite, in an enzymatic
step that is effectively irreversible (Figure
1). While anabolism is clearly critical in the conversion of
5-FU to the active nucleotides FdUMP, FUTP, and FdUTP (these
metabolites can in turn inhibit cell replication through inhibition
of thymidylate synthase, or through incorporation into RNA or DNA,
respectively), catabolism controls the amount of 5-FU available for
anabolism and thus occupies a critical position in the overall
metabolism of 5-FU.
Because of the widespread use of fluoropyrimidines in oncology, it is
particularly desirable to be able to assess DPD activity in the
clinical setting. The ability to measure DPD in human tissue has
further increased our appreciation of the importance of DPD for 5-FU
pharmacology (see below).
Currently there are three different methods used to measure DPD
activity. Two of the methods are direct assays of DPD activity; one
being an HPLC-radioassay for DPD activity and the second being an
immunoblot assay of DPD protein. A third method, useful mainly in
the in vivo or clinical setting, is an indirect assessment of DPD
activity. By quantitating the levels of uracil, the natural substrate
for DPD that increases when DPD is inhibited, an estimate of
remaining DPD activity can be made.
For most clinical studies of DPD activity, peripheral blood
mononuclear cells, isolated from heparinized blood on Ficoll-hypaque,
can be used to monitor for DPD activity. Following preparation of
cytosol, DPD activity can be assayed as follows: At a specific
protein concentration of cytosol, incubation is carried out at
37°C in the presence of reduced nicotinamide-adenine
dinucleotide phosphate (NADPH) and radiolabeled 5-FU. Samples can
then be removed at specified times (over 30 min) and analyzed by
reversed phase HPLC using a radiodetector. DPD activity is then
expressed as nmol of 5-FUH2 formed per min per mg of protein. This is
described more fully elsewhere.
The importance of DPD to the clinical pharmacology of 5-FU has been
shown in several recent studies that demonstrate how DPD can
influence the pharmacokinetics, bioavailability, toxicity, and
antitumor effectiveness of 5-FU.
DPD is known to have a circadian pattern in both animals and
humans.[6-8] Studies in patients given 5-FU infusion by automated
pumps have demonstrated that the circadian variation of tissue DPD
level is accompanied by an inverse circadian pattern in plasma 5-FU
concentrations. This has potential importance in the design of
time-modified 5-FU infusions. Such regimens have been suggested to
have potential benefit in the treatment of certain human cancers.
DPD enzyme activity in normal tissues (peripheral blood mononuclear
cells and liver) has also been shown to vary among individuals in a
Gaussian pattern, with as much as a sixfold variation from the lowest
to the highest values.[10,11] This wide variation in DPD activity is
likely responsible for the wide variation in the t½b observed in
patients in population studies.
In addition to the variation of DPD activity observed in the normal
population, it is now clear that an additional small percentage (<
5%) of the population has DPD activity significantly below the
Gaussian distribution that characterizes most of the
population.[13-15] These individuals are at significant risk if they
develop cancer and are given 5-FU. This is a true pharmacogenetic
syndrome with symptoms that are not recognized until patients are
exposed to the drug.
Recently, variation in DPD activity has also been shown to be
responsible for variable bioavailability observed following oral
administration of 5-FU. The erratic bioavailability of 5-FU had not
previously been understood, particularly because 5-FU is a relatively
small molecule with a pKa that would predict excellent absorption and
bioavailability. Experimental studies using DPD inhibitors have
demonstrated in rodents that following inhibition of DPD the
pharmacokinetic pattern resulting from oral administration of 5-FU is
essentially the same as that produced by intravenous administration,
suggesting almost 100% bioavailability.
Tumors may also express a variable level of DPD activity. This
potentially may explain the observed varied tumor response to
5-FU. Tumors with relatively low levels of DPD should predict
sensitivity to 5-FU, while tumors with relatively high levels of DPD
should predict resistance to 5-FU.
The studies described above detailing the variability in DPD levels
in both normal and tumor tissues provide an explanation for the
observed variability in 5-FU pharmacology and at the same time
suggest a potential target for chemotherapy. It thus becomes
attractive for the oncologist to consider inhibiting DPD in order to
eradicate the variability in 5-FU pharmacology. Inhibiting DPD in 5-FU-susceptible
host tissue, such as GI mucosa and bone marrow, should make dosing
from patient to patient less variable. Inhibition of DPD in tumor
specimens is also attractive because it is likely that many tumors
achieve resistance to 5-FU through an increase in DPD activity within
the tumor resulting in increased degradation and thus less
opportunity for anabolism of 5-FU.
Over the years there have been many attempts to synthesize effective
inhibitors of DPD. Unfortunately, many of these compounds have
proven to be very toxic. In the past several years, new
fluoropyrimidine drugs introduced into the clinic have shown that
pharmacologic modulation of DPD can result in antitumor efficacy with
Four new fluoropyrimidine drugs using DPD modulators are currently
being evaluated in clinical studies. These include UFT, eniluracil,
S-1, and BOF-A2. These drugs differ in the type of DPD modulation as
well as the relative degree of inhibition of DPD produced. All of
these drugs achieve therapeutic gains from DPD modulation. Most
impressive is the fact that DPD modulation permits oral
administration of these drugs and results in less pharmacokinetic variability.
UFT was the first of these drugs to be synthesized (more than 20
years ago) and is the one for which we have the most clinical
experience. This novel fluoropyrimidine is a combination of the
naturally occurring pyrimidine uracil with the fluoropyrimidine
tegafur (ftorafur) in a 4:1 molar ratio. The presence of uracil in
excess of 5-FU results in competition at the level of DPD such that
5-FU formed from tegafur will not be rapidly degraded and will remain
present for a prolonged period (Figure
2). While not actual inhibition of DPD, the competition at the
DPD level produces an effect similar to that accomplished with a true
DPD inhibitor. In contrast to true DPD inhibitors and inactivators,
the effect on DPD is more rapidly reversible. There are now extensive
data from Japan as well as Europe, South America, and the United
States demonstrating that oral UFT administered either as a single
agent or combined with leucovorin has antitumor activity in several
tumor types (particularly breast and colon cancer) that is at least
as good as that achieved with IV 5-FU. Furthermore, the toxicity
profile has been shown to be tolerable, with the typical
fluoropyrimidine toxicities (eg, diarrhea and nausea) seen at the
maximal tolerated dose (MTD). Of note is the virtual absence of other
toxicities, in particular hand-foot syndrome, neurologic toxicity,
and cardiotoxicity. Although not well understood, these
toxicities have been thought to be possibly secondary to the
formation of 5-FU catabolites. Because of modulation at the DPD
level, 5-FU catabolites are less likely to form from UFT.
In Japan, there have been several attempts to develop this drug
combination concept further. S-1 is a triple drug combination (Figure
3) consisting of the prodrug tegafur (same as in UFT) together
with a true DPD inhibitor 5-chloro-2,4-dihydroxypyridine (CDHP) and
potassium oxonate in a molar ratio of 1:0.4:1, respectively. S-1
permits sustained 5-FU release from the 5-FU prodrug, with DPD
modulation in this combination being more potent secondary to the
effect of an actual DPD inhibitor. The third drug in this
combination, potassium oxonate, was added theoretically to lessen the
potential for typical 5-FU gastrointestinal toxicity (particularly
diarrhea) seen with most fluoropyrimidine drugs. In preclinical
studies, potassium oxonate was shown to selectively inhibit 5-FU
phosphorylation by the enzyme orotate phosphoribosyltransferase,
particularly in the gastrointestinal tract but not in tumors.
Results of preclinical studies have been encouraging, demonstrating
excellent antitumor activity. In clinical studies thus far, S-1
has been quite tolerable.[27,28]
BOF-A2 represents another attempt to develop an improved
fluoropyrimidine drug. With this two-drug combination, the prodrug
1-ethoxymethyl 5-fluorouracil (EM-FU) is combined with the DPD
inhibitor 3-cyano-2,6-dihydroxypyridine (CNDP) in a 1:1 molar
ratio. EM-FU is relatively resistant to degradation and is
metabolized to 5-FU by the liver microsomes. Preclinical studies have
confirmed the antitumor activity of BOF-A2 in several animal models
and have also demonstrated sustained 5-FU levels resulting from
release of 5-FU from EM-FU. Clinical studies have been conducted in
Japan, and more recently, limited studies have begun in the United
States. It is too early to comment on the possible clinical
effectiveness of this drug combination because of the limited patient
data available. In US studies of BOF-A2 thus far, however, typical
5-FU toxicities were observed, with some patients experiencing more
severe toxicity. At present, the dose, schedule, and potential
for combining this agent with other modulators (eg, leucovorin) are
Recently, eniluracil (ethynyluracil or GW776C85), an even more potent
inhibitor of DPD, has been synthesized. This agent has been
demonstrated to be a potent inactivator of DPD.
Eniluracil is a pyrimidine with a structure similar to that of both
uracil and 5-FU (Figure 4) that
has been shown to rapidly and completely inactivate DPD (Figure
5). Animals exposed over prolonged periods to relatively low
doses of eniluracil alone had no obvious toxicity. Following the
inhibition of DPD in these animals, a concomitant increase in plasma
uracil has been observed. Identification of an effective,
nontoxic dose of eniluracil has permitted evaluation of this drug
combined with various low doses of 5-FU. Results of pharmacokinetic
studies in rodents demonstrated remarkably reproducible 5-FU
pharmacokinetics. Subsequent rodent studies showed that 5-FU
could be administered orally in reproducible intra-animal and
interanimal studies, with bioavailability being demonstrated to be
The effectiveness of 5-FU and eniluracil in inhibiting tumor growth
has been demonstrated in several animal models, with evidence of
complete tumor regression in models in which only modest antitumor
effect had previously been seen.
Initial phase I clinical studies with eniluracil examined
administration of the drug alone orally for 7 days at doses of 0.74,
3.7, or 18.5 mg/m² to determine both the initial clinical
pharmacologic characteristics and the toxicity profile.
Pharmacokinetic evaluation demonstrated a t½ of 4.5 hours for
each of these doses. No changes were noted in the pharmacokinetics of
eniluracil with repeated doses compared with single doses.
Of particular interest is the effect of these doses of eniluracil on
DPD activity in the clinical setting. To assess DPD activity,
peripheral blood mononuclear cells were examined using the
methodology described above. Results demonstrated that DPD was
rapidly and completely inactivated by eniluracil, and inhibition was
maintained for more than 1 day at clinically used doses (Figure
6). Currently, phase II studies are ongoing or planned to
evaluate the effectiveness of the coadministration of 5-FU and
eniluracil in several different malignancies.
Improved efficacy of chemotherapy may result from eliminating 5-FU
degradation by tumor DPD, making DPD inactivation appealing as a
therapeutic goal. Studies are under way to evaluate whether
eniluracil can produce this effect in clinical studies. One
concern, however, is the length of time DPD remains inhibited in
normal host tissues, and clinical studies are currently planned to
evaluate this potential toxicity.
In summary, DPD activity is a critical step in pyrimidine metabolism
and is responsible for much of the variability in pharmacokinetics,
oral bioavailability, toxicity, and efficacy following administration
of 5-FU. Modulation of DPD activity through relative DPD inhibition
should result in less variation in 5-FU pharmacokinetics and
bioavailability and potentially may improve the drugs
therapeutic effectiveness both by making toxicity (after 5-FU dosing)
more predictable and by overcoming the high levels of DPD activity in
tumors where elevated DPD is a mechanism of tumor resistance. The
recent availability of DPD modulators such as UFT, S-1, BOF-A2, and
eniluracil offer the potential to decrease the variability due to DPD
and produce an improved fluoropyrimidine therapeutic effect. The type
of DPD modulation and relative degree of inhibition of DPD varies
with each drug. These differences may in turn account for differences
in efficacy and toxicity. Further investigation, including
comparative studies, is needed to clarify these differences.
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