Clinical Implications of Dihydropyrimidine Dehydrogenase on 5-FU Pharmacology

ABSTRACT: Dihydropyrimidine dehydrogenase (DPD) is the initialrate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU), accounting forcatabolism of over 85% of an administered dose of 5-FU. DPD plays an importantrole in regulating the availability of 5-FU for anabolism. DPD also accounts formuch of the variability observed with the therapeutic use of 5-FU. This includesvariable 5-FU levels over 24 hours during a continuous infusion; the widelyreported variability in the pharmacokinetics of 5-FU; the observed variablebioavailability that led to the recommendation that 5-FU not be administered asan oral agent; and lastly, the observed variability in both toxicity and drugresponse (resistance) after identical 5-FU doses. Knowledge of the DPD level, aswell as the levels of other potentially important molecular markers (eg,thymidylate synthase), may permit adjustments or modulation of the 5-FU dosethat can result in an increase in the therapeutic efficacy of 5-FU. [ONCOLOGY 15(Suppl2):21-27, 2001]

Introduction

Dihydropyrimidine dehydrogenase (also known as DPD,dihydrouracil dehydrogenase, dihydrothymine dehydrogenase, uracil reductase, EC1.3,1.2) is the initial rate-limiting enzymatic step in the catabolism of notonly the naturally occurring pyrimidines, uracil and thymine, but also thewidely used antimetabolite cancer chemotherapy drug, 5-fluorouracil (5-FU).[1,2]As shown in Figure 1, DPD occupies an important position in the overallmetabolism of 5-FU, converting over 85% of a standard dose of administered 5-FUto dihydrofluorouracil (5-FUH₂), an inactive metabolite, in an enzymatic stepthat is essentially irreversible.[3]

It is true that anabolism is clearly critical for 5-FU cytotoxicaction through conversion of 5-FU to the "active" nucleotides5-fluorodeoxyuridine monophosphate (FdUMP), 5-fluorouridine triphosphate (FUTP),and 5-fluorodeoxyuridine triphosphate (FdUTP). These important activemetabolites are, in turn, responsible for inhibition of cell replication throughinhibition of thymidylate synthase, or through incorporation into RNA or DNA,respectively. Nevertheless, it is pyrimidine catabolism through therate-limiting step, DPD, that controls the availability of 5-FU for anabolismand thus occupies a critical position in the overall metabolism of 5-FU.

Evidence of Importance of DPD Activity to 5-FU Pharmacology

DPD—as the rate-limiting step in pyrimidine catabolism—hasrecently been shown to play a critical role in determining the clinicalpharmacology of 5-FU (Table 1). In particular, it has been demonstrated that DPDaccounts for much of the variability that has been noted in clinical studieswith 5-FU. This includes both intrapatient variability, as well as interpatientvariability.

DPD activity has been observed to follow a circadian pattern inboth animals and humans.[4-6] Studies in rats on a 12-hour light/12-hour darkschedule have demonstrated that hepatic levels of DPD follow a pattern that canbe plotted on a cosine wave.[4] This pattern was completely reversed in anothergroup of rats on an inverted 12-hour light/12-hour dark schedule. In patientsreceiving continuous-infusion 5-FU by automated pumps, sampling DPD activity inperipheral blood mononuclear cells over a 24-hour period has also been shown toexhibit circadian patterns when plotted on a cosine wave.[6] Serum samplesobtained at the same time from the same patients have been shown to have serum5-FU concentrations that were also characterized by a circadian pattern, whichwas essentially inverse to the DPD circadian pattern (Figure2).

Data from this study suggested that perhaps DPD was responsiblefor the circadian variation in 5-FU, leading some chemotherapists to proposetime-modified 5-FU infusions to optimize drug delivery during a 24-hour period.Such regimens have been suggested by some oncologists, particularly those inWestern Europe, to have a potential benefit in the treatment of certain humancancers.[7]

What Causes the Variation?

For the past 4 decades, it has been unclear as to why thepharmacokinetics of 5-FU have been so variable, with half lives (t_1/2) rangingfrom around 4 minutes to 25 minutes after an intravenous bolus (Table2).[8]Because of the critical position of DPD in the metabolic pathway, it washypothesized that the variation in pharmacokinetic characteristics might besecondary to variability of DPD enzyme activity among different individuals.Population studies were undertaken assessing DPD enzyme activity initially intissues (peripheral blood mononuclear cells and liver) from healthy individuals.DPD was shown to vary from individual to individual, with a normal distributionpattern (bell-shaped curve) showing a six-fold variation from the lowest to thehighest values (Figure 3).[9,10]

Essentially the same pattern of DPD activity has also beenobserved in the peripheral blood mononuclear cells of both breast and colorectalcancer patients, although, interestingly, the normal distribution is shifted tothe left, with lower median DPD activity in the breast cancer patientpopulation.[11] The mechanism for this latter observation is unknown. The widevariation in DPD activity observed in the populations described above is thoughtto account for the wide variation in the half-life (and pharmacokinetics)observed in patients treated with 5-FU.[8]

Variation in DPD Activity

While most patients tolerate 5-FU reasonably well, over the past4 decades, a number of patients have developed severe, at timeslife-threatening, toxicity after standard doses of 5-FU.[12-15] Because thesepatients demonstrated exaggerated normal toxic side effects (as if they hadreceived an overdose of drug), it was hypothesized that these patients weredeficient in a catabolic enzyme that resulted in more 5-FU being present overtime.

Initial studies in these affected patients demonstrated elevateduracil and thymine levels, suggesting a deficiency in the first step inpyrimidine catabolism, DPD.[12-14] Subsequent studies demonstrated that many ofthese patients were indeed DPD deficient. It is now clear that an additionalsmall percentage (< 3%) of such patients have DPD activity that issignificantly below the normal distribution seen in the average person.[15]These individuals are at significant risk if they develop cancer and are given5-FU. This is a true pharmacogenetic syndrome, with symptoms beingunrecognizable until exposure to the drug.[15]

Variation in DPD activity has also recently been shown to beresponsible for the apparent variable bioavailability of 5-FU, which over theyears has led to the recommendation that 5-FU not be administered by the oralroute. An explanation for the erratic bioavailability of 5-FU has previouslybeen unclear, particularly since 5-FU is a small molecule with a pK_a that shouldfavor excellent absorption and bioavailability. Recent studies using DPDinhibitors in animals have demonstrated that, following inhibition of DPD, thepharmacokinetic pattern resulting from oral administration of 5-FU isessentially the same as that produced by intravenous administration, resultingin essentially 100% bioavailability.[16]

While it has long been well known that variability in theactivity of pyrimidine anabolic enzymes is important in determining theantitumor effectiveness of 5-FU, much less attention has focused on thevariability in activity of the pyrimidine catabolic enzymes. Tumors that areresistant to 5-FU have been shown to express increased levels of DPDactivity.[17] With the development of the quantitative polymerase chain reaction(PCR) to measure DPD mRNA, increased expression of DPD mRNA has beendemonstrated in tumors of patients who were resistant to 5-FU.[18]

Developing a Pharmacologic Strategy Based on DPD

The variability in DPD levels in both normal and tumor tissuesprovides a basis for the chemotherapist to consider either altering the dose ofthe fluoropyrimidine drug or inhibiting DPD in order to eradicate thevariability in 5-FU pharmacology. The presence of increased tumor DPD could leadto a decision to increase the dose of 5-FU (or a 5-FU prodrug) to overcome theincreased catabolism of 5-FU within the tumor, thus increasing the amount of5-FU that could be converted to active anabolites (Figure1).

An alternative approach is to use known inhibitors of DPD,usually with a lower dose of 5-FU (or the use of a 5-FU prodrug; eg,capecitabine [Xeloda]), to directly inhibit 5-FU degradation within the tumor,thereby permitting the existing 5-FU (even if present in low concentrations) tobe converted to active anabolites. Inhibiting DPD in 5-FU-susceptible hosttissue, such as gastrointestinal mucosa and bone marrow, should also make dosingfrom patient to patient less variable, the latter being accomplished throughlessening the variability in pharmacokinetics, bioavailability, and theresultant host toxicity.

Dihydropyrimidine Dehydrogenase Inhibitory Fluoropyrimidines

Over the years, there have been many attempts to synthesizeeffective inhibitors of DPD.[19] Unfortunately, many of these compounds haveproven to be very toxic. In the past several years, several fluoropyrimidinedrugs using DPD inhibition have been introduced into the clinic. These drugs,referred to as dihydropyrimidine dehydrogenase inhibitory fluoropyrimidines(DIF), include uracil and tegafur (UFT), eniluracil (GW-776C85;5-ethynyluracil), S-1, and emitefur (BOF-A2), all of which have recently been inclinical studies.[20] These drugs differ both in type of DPD"inhibition," as well as degree of inhibition produced.

The rationale for using DIF drugs is that they are a source of5-FU, either from 5-FU itself or from a "prodrug" that is converted to5-FU, combined with another agent that interferes with (or inhibits) theotherwise rapid catabolism of 5-FU. This permits oral delivery of 5-FU(bioavailability > 70%) and results in less variability in thepharmacokinetics of the fluoropyrimidines. In addition, by inhibiting thecatabolic pathway, more 5-FU can enter the anabolic pathway, thereby potentiallyincreasing the antitumor effect. This is theoretically important for tumors thatare resistant secondary to an increase of intratumoral DPD. Lastly, while notcompletely understood, it is thought that some 5-FU toxicities (hand-footsyndrome; some forms of neurotoxicity; and possibly, cardiotoxicity) may besecondary to the catabolic pathway. Inhibiting the catabolic pathway mightdecrease the incidence of these toxicities.

UFT—The Most Studied Dehydrogenase Inhibitory Fluoropyrimidine

UFT was the first DIF drug to be synthesized; it is also themost studied DIF drug.[21,22] This "new" fluoropyrimidine is acombination of the naturally occurring pyrimidine, uracil, with thefluoropyrimidine, tegafur (ftorafur), in a 4:1 molar ratio. The presence ofuracil in excess of 5-FU results in "competition" at the level of DPD,such that the 5-FU, which is formed from tegafur, will not be rapidly degradedand will remain present for a prolonged period. While this is not a true"inhibition" of DPD, the competition between 5-FU and uracil for DPDproduces an effect similar to that achieved with a true DPD inhibitor.

In contrast to the true DPD inhibitors and inactivators, theeffect on DPD is more rapidly reversible. This rapidly reversible inhibition mayavoid some of the problems seen with the earlier DPD inhibitors, and may alsoaccount for a more favorable toxicity profile compared to some of the earlierDPD inhibitors,[19] as well as some of the newer DIF drugs.

Extensive data from Japan, as well as Europe, South America, andthe US, now demonstrate that orally administered UFT has antitumor activity inseveral tumor types (particularly breast and colon cancer), either as a singleagent or combined with leucovorin.[23] Studies thus far have shown that it is atleast as effective as intravenously infused 5-FU. Furthermore, its toxicityprofile has proven quite tolerable, with the typical fluoropyrimidine toxicities(eg, diarrhea and nausea) seen at the maximum-tolerated dose. Of note is thepaucity of other toxicities, in particular hand-foot syndrome, neurologic, andcardiotoxicity.[24] Although not well understood, these toxicities may besecondary to 5-FU catabolites. Such catabolites are less likely to form fromUFT; therefore, these toxicities are not typically observed.

Conclusion

While DPD is clearly an important factor,[25] a more rationalapproach would probably be to monitor multiple factors that are known to predict5-FU effectiveness. These might include not only DPD, but also the targetthymidylate synthase, and possibly the enzymatic steps leading to anabolism of5-FU to active nucleotide anabolites. One could then base the modulation of 5-FUon the total picture. In particular, immunohistochemical evaluation orquantitative PCR of multiple targets may provide a valuable approach to suchassessment.

Recently, patients in a clinical trial for advanced colorectalcancer were assessed for response to 5-FU plus leucovorin (Mayo Clinic Regimen)and independently assessed for levels of DPD, thymidylate synthase, andthymidine phosphorylase. Such assessment proved quite accurate in predictingresponse to therapy. Patients with relatively "low" expression of allof these markers responded to the regimen, while those patients who showedelevation of even one marker were unresponsive (Figure4). It is likely thatthis type of approach, using assessment of multiple markers, will become thestandard of care in the future.

Questions and Answers

Daniel Haller, MD: This questions concerns dihydropyrimidinedehydrogenase (DPD) activity and Dr. Diasio’s statement that the normaldistribution is shifted to the left, with lower median DPD activity in thebreast cancer population. Is the breast cancer effect one of gender or is it thetype of tumor?

Robert Diasio, MD: No, it is not the gender effect. As a matterof fact, we have taken a corresponding group of similar age colorectal cancerpatients and head and neck cancer patients who were getting 5-FU and we do notfind it. We do not understand what it is due to, but there is a definite shift.We have studied more than 500 breast cancer patients in our population and wehave looked at other factors like menopausal status, adjuvant setting vsadvanced disease, but we do not have any good explanation of any factor that isresponsible for it. But there is a definite shift to the left of the normaldistribution.

Dr. Haller: If DPD deficiency is seen in 5% to 6% of people, whydo we see it so rarely in clinical practice? Is there an artificial cutoffpoint?

Dr. Diasio: I think that it is relative. We have seen it in anumber of patients. The 6% figure I use for breast cancer is based on somewherebetween 500 and 600 patients—all of the breast cancer patients who come to ourclinic. Some of those patients were not getting 5-FU therapy. Others were. Wecontinue to be impressed that though this is not a common problem, it is outthere, and there are more patients who have this than I think we ever previouslythought existed.

Dr. Haller: How many patients have you documented with lethaltoxicity?

Dr. Diasio: Seven lethal patients. Those seven patients diedfollowing administration of chemotherapy, several of them after the first cycleof chemotherapy where they became septic. But we have a large percentage ofpatients who are heterozygotes and have only one defect. There are at least 13different mutations that have been documented now.

The 5% to 6% DPD deficiency is specifically for breast cancer.With colorectal cancer, it is about 3% of the patients that we have found.

John Marshall, MD: Would it make sense to use a DPD inhibitorwith capecitabine?

Dr. Diasio: It is something that has been proposed by severalinvestigators and is being considered. I think at this point there is more focusreally on the use of molecular markers to characterize the tumors. It issomething that could be considered in the future. I think that a lot of thetoxicities we feel are related to the catabolic pathway probably can be dealtwith much in the way we deal with the Lokich infusional 5-FU regimen and thatis, by turning down the amount of 5-FU either by pausing or reducing the dose.

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