In the presence of NADPH, dihydropryimidine dehydrogenase (DPD) catalyzes the rate-limiting step in the catabolism of pyrimidine bases, including uracil, thymine, and the anticancer agent 5-fluorouracil (5-FU; Fig. 1). Following bolus and continuous infusion administration of 5-FU, about 90% and 99% of the drug is eliminated by catabolism, respectively, whereas renal excretion of parent drug accounts for the balance (1, 2). This enzyme therefore indirectly influences the amount of 5-FU available for anabolism to the active nucleotide metabolites, 5-fluoro-2′-dUMP, which inhibits thymidylate synthase, and 5-fluorouridine triphosphate, which incorporates extensively into RNA. Saturation of DPD at drug concentrations several-fold above the affinity constant accounts for the nonlinear pharmacokinetics of 5-FU.

Fig. 1.

Anabolism and catabolism of 5-FU. Abbreviations: F(d)Urd, 5-fluoro(2′-deoxy)uridine; F(d)UMP, 5-fluoro(2′-deoxy)uridine monophosphate; F(d)UDP, 5-fluoro(2′-deoxy)uridine diphosphate; F(d)UTP, 5-fluoro(2′-deoxy)uridine triphosphate; DHFU, dihydrofluorouracil; FBAL, fluoro-β-alanine; TS, thymidylate synthase.

Fig. 1.

Anabolism and catabolism of 5-FU. Abbreviations: F(d)Urd, 5-fluoro(2′-deoxy)uridine; F(d)UMP, 5-fluoro(2′-deoxy)uridine monophosphate; F(d)UDP, 5-fluoro(2′-deoxy)uridine diphosphate; F(d)UTP, 5-fluoro(2′-deoxy)uridine triphosphate; DHFU, dihydrofluorouracil; FBAL, fluoro-β-alanine; TS, thymidylate synthase.

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In this issue, the results of a population-based study of DPD activity in peripheral blood mononuclear cells obtained from 150 healthy Japanese volunteers is reported by Ogura et al. (3). Consistent with other population studies, a Gaussian distribution was observed, with a large degree of interindividual variation. The authors report that the mean activity was 0.173 nmol/min/mg, and the ratio of maximum to minimum values was 26.5. Further evaluation of DPD protein content and mutational analysis was pursued in the patient with the lowest activity (8% of the mean). Immunoblot analysis confirmed a trace amount of protein expression. Reverse transcription-PCR analysis of the coding region of DPD cDNA revealed two novel substitutions: 1097G > C and 2303C > A, that resulted in amino acid substitutions G366A and T768K, respectively. The mutant types of DPD were expressed in Escherichia coli and purified. DPD containing the G366A mutation had a markedly reduced affinity toward NADPH, but normal affinity to 5-FU, with a reduction in the kinetic variables. The T768K mutation did not affect the affinity to NADPH or the kinetic variables but did influence thermal stability. The authors are to be complemented on this elegant study.

The incidence and type of grade 3 to 4 toxicities is influenced by the dose and schedule of 5-FU. For example, a meta-analysis comparing bolus administration with continuous infusion 5-FU revealed a 7.8-fold higher incidence of hematologic toxicity with bolus 5-FU (31% versus 4%), whereas hand-foot syndrome was 2.6-fold higher with the infusional schedules (34% versus 13%; ref. 4). Although not all patients who experience serious toxicity with 5-FU are DPD deficient, profound DPD deficiency has been identified in a subset of patients who have experienced excessive toxicity with 5-FU-based therapy. Prospective identification of patients with an increased risk of developing life-threatening or fatal 5-FU-associated toxicity would obviously be useful, so that either significant dose modification or the use of non-5-fluoropyrimidine drugs could be employed. Complete DPD deficiency is estimated to occur in about 0.1% of patients, whereas relative DPD deficiency is projected to have an incidence of 3% to 5% (4). Therefore, an important clinical issue concerns whether screening of patients before receiving 5-FU or 5-FU prodrugs is feasible and cost-effective. DPD enzymatic activity is typically measured by incubating cellular lysates at 37°C with excess NADPH and radiolabeled 5-FU, with separation of 5-FU and its catabolites by high-performance liquid chromatography. This is a labor-intensive assay, which has limited its availability in general clinical laboratories. The activity in peripheral blood mononuclear cells is influenced by whether the assay is done on fresh or frozen samples, and activity may vary within individuals throughout the day, which poses logistical problems in establishing reference assays to identify subjects with partial DPD deficiency. Are there other options?

A reduced “test” dose might allow a physician to see whether a patient has exquisite sensitivity to 5-FU, but because the onset of symptoms may be delayed, this may not be practical, particularly for newly diagnosed patients. Pharmacokinetic monitoring following a test dose would make this approach more useful, but this approach is not currently available outside the research laboratory setting.

Serious clinical toxicity tends to increase with higher 5-FU systemic exposure, reflected by total area under the plasma-concentration time curve with bolus injection and steady-state plasma concentrations with 5-FU infusion. Some clinical trials involving infusional 5-FU-based regimens suggest that pharmacokinetic monitoring can be employed to adjust 5-FU doses to avoid or minimize serious clinical toxicity, but this is also a labor-intensive approach (5).

Measuring uracil levels in plasma or urine represents an alternate approach. For example, in a study involving 5-FU given with the DPD inhibitor eniluracil, plasma uracil levels >0.95 μmol/L (measured by gas chromatography-mass spectroscopy) were associated with significantly lower DPD activity (6). Other investigators have proposed that the ratio of 5,6-dihydrouracil to uracil in plasma reflects DPD activity and may be useful in predicting toxicity of 5-FU-based therapy (7).

Pharmacogenomics represents another method for screening. The structural organization of the human DPD gene indicates that it is a single copy gene on chromosome 1p22 and consists of 23 exons. Several dozen different mutations and polymorphisms have been identified in the DPD gene, and most of these have been detected in patients with profound DPD deficiency (8). Mutations have been identified in both introns and exons and include point mutations, splice site mutations, frame-shift mutations, nonsense mutations, and deletions due to exon skipping. The frequency of these mutations may vary among different ethnic populations.

Mattison et al. recently reported the potential of using a novel 2-13C-carbon-uracil breath test as a means of identifying DPD deficiency (9). These authors studied 50 normal subjects and eight individuals with either partial (n = 7) or profound (n = 1) DPD deficiency. Patients ingested an aqueous solution of 2-13C-uracil, and radioactive carbon dioxide levels (see Fig. 1) in exhaled breath were determined at various intervals by a tabletop IR spectroscopy instrument. The data suggested that a single time point determination was able to discriminate between subjects with normal activity and DPD deficiency; importantly, the test also seemed to distinguish those with partial and profound DPD deficiency. This method offers the potential for office or hospital-based testing. To date, however, there have been no large, prospective clinical trials to test whether such screening of DPD activity will result in a benefit in terms of reduced toxicity. With the potential availability of a simple screening test, however, clinical trials evaluating the worth of prospective testing may now be feasible.

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