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Phase I and Pharmacokinetic Trial of Weekly Oral Fluorouracil Given With Eniluracil and Low-Dose Leucovorin to Patients With Solid Tumors

From the Medicine Branch, Division of Clinical Sciences, National Cancer Institute, National Naval Medical Center; Hematology/Oncology Section, Department of Internal Medicine, National Naval Medical Center; and Department of Radiology, National Naval Medical Center, Bethesda, MD.

Address reprint requests to Jean Grem, MD, National Cancer Institute–Medicine Branch, National Naval Medical Center, 8901 Wisconsin Ave, Bldg 8, Rm 5101, Bethesda, MD 20889; email jgrem{at}helix.nih.gov

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: Fluorouracil (5-FU) given as a weekly, high-dose 24-hour infusion is active and tolerable. We evaluated an oral regimen of eniluracil (which inactivates dihydropyrimidine dehydrogenase [DPD]), 5-FU, and leucovorin to simulate this schedule.

PATIENTS AND METHODS: Patients received a single 24-hour infusion of 5-FU (2,300 mg/m² on day 2) with leucovorin (15 mg orally [PO] bid on days 1 through 3) to provide reference pharmacokinetic data. Two weeks later, patients began treatment with eniluracil (20 mg) and leucovorin (15 mg) (PO bid on days 1 through 3) and 5-FU (10 to 15 mg/m² PO bid on day 2).

RESULTS: Dose-limiting toxicity (diarrhea, neutropenia, and fatigue) was seen with 5-FU 15 mg/m² PO bid on day 2 given weekly for either 6 of 8 weeks or 3 of 4 weeks, whereas five of seven patients tolerated 5-FU 10 mg/m² PO bid given weekly for 3 of 4 weeks. Eniluracil led to a 35-fold reduction in 5-FU clearance. Fluoro-beta-alanine, a 5-FU catabolite, was not detected in plasma during oral 5-FU–eniluracil therapy. DPD activity was markedly suppressed in all patients during eniluracil therapy; the inactivation persisted after the last eniluracil dose; percentages of baseline values were 1.8% on day 5, 4.5% on day 12, and 23.6% on day 19.

CONCLUSION: The recommended oral dosage of 5-FU (10 mg/m² PO bid) given with eniluracil and leucovorin is approximately 115-fold lower than the reference dosage for 24-hour infusional 5-FU. This difference is greater than expected given the reduction in 5-FU clearance. DPD inactivation persisted for several weeks after completion of eniluracil therapy.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PRECLINICAL AND clinical studies have shown that both fluorouracil (5-FU) concentration and duration of exposure are critical determinants of cytotoxicity.¹ Several randomized trials have demonstrated that administration of single-agent 5-FU by protracted continuous intravenous (IV) infusion has an advantage over bolus injection in terms of response rate and toxicity profile.^2-5 A meta-analysis of six randomized trials confirmed the superior response rate associated with infusional 5-FU and revealed a small but statistically significant survival advantage.⁶ A variety of infusion schedules have been developed, but there is no consensus regarding the optimal regimen. Ardalan et al⁷ first evaluated the administration of high-dose 5-FU by weekly infusion over 24 hours. They and others documented that this was an active schedule that provided a high 5-FU dose-intensity but with a favorable toxicity profile.^7-10 Gastrointestinal toxicity and myelosuppression are manageable, although neurotoxicity characterized by symptoms of cerebellar ataxia may be dose limiting. Randomized studies have shown that the weekly 24-hour infusion schedule with or without leucovorin modulation is less toxic than modulated bolus 5-FU regimens.^3,11,12

Oral administration of 5-FU was previously ruled out because of low and erratic bioavailability, due to first-pass catabolism of 5-FU by dihydropyrimidine dehydrogenase (DPD) in both the intestinal mucosa and the liver. A number of strategies have been developed to permit oral administration of 5-FU to mimic various continuous infusion schedules without the need for indwelling IV catheters and use of an infusion pump. Eniluracil is a potent mechanism-based inactivator of DPD.¹³ Both preclinical and clinical studies have shown that eniluracil administration results in complete inactivation of DPD, as evidenced directly by enzyme assays and indirectly by markedly increased plasma uracil levels.^14-19 Oral administration of 5-FU with eniluracil is associated with essentially 100% bioavailability, and renal excretion is the predominant form of elimination of 5-FU in this case. An attractive feature of eniluracil therapy is the potential to prevent the formation of catabolites of 5-FU, which may contribute to host toxicity and interfere with 5-FU cytotoxicity.^20-23 Further, eniluracil prevents the neurotoxicity that has been associated with a 72-hour infusion of 5-FU in dogs.²⁴ Laboratory and clinical evidence suggests that increased expression of DPD in tumor tissue may be associated with insensitivity to 5-FU.^25-28 A 3-day course of twice-daily eniluracil has been shown to inactivate DPD completely in primary tumor tissue of colorectal cancer patients,²⁹ which suggests that eniluracil may circumvent a potential resistance mechanism.

We wished to develop a weekly oral regimen of eniluracil, 5-FU, and leucovorin that would simulate a weekly, high-dose, 24-hour infusion schedule. Fixed doses of eniluracil and leucovorin were to be given twice daily on days 1 through 3, with oral 5-FU given twice on day 2. The goals of this study were to define a tolerable dose of 5-FU, to compare the pharmacokinetic profile of a 24-hour infusion of 5-FU (with oral leucovorin on days 1 through 3) and oral 5-FU (given with eniluracil and leucovorin on days 1 through 3), and to monitor DPD activity during and after eniluracil therapy.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Eligibility
This study was activated in January 1998, accrual was completed in October 1998, and the last remaining patient was removed from protocol therapy in June 1999. Patients with solid tumors for whom a 5-FU–leucovorin–based regimen represented a reasonable therapeutic approach or for whom no effective standard therapy was available were eligible. There were no restrictions with regard to prior chemotherapy. Patients were required to have an Eastern Cooperative Oncology Group performance status of 2 or better and adequate organ function (granulocyte count > 2,000/µL, platelet count > 100,000/µL, bilirubin level < 2.0 mg/dL, AST level < four times normal, creatinine level < 1.6 mg/dL, and creatinine clearance > 55 mL/min). This study (FUMA5008) had the approval of the local institutional review boards and the Cancer Therapy Evaluation Program (CTEP), National Cancer Institute; all patients gave written informed consent.

Treatment Plan
Oral tablets of eniluracil (10 mg) and 5-FU (5 and 25 mg) were formulated by Glaxo Wellcome (Research Triangle Park, NC) and supplied by CTEP. Commercial sources were used for the IV 5-FU and oral leucovorin. During the initial period, leucovorin 15 mg orally (PO) was given twice daily on days 1 through 3. On day 2, 5-FU 2,300 mg/m² was given by continuous infusion over a 24-hour period. The patient returned 2 weeks later to commence treatment with eniluracil (20 mg) and leucovorin (15 mg) (PO bid on days 1 through 3). 5-FU was given PO 12 hours apart on day 2; the starting dose was 15 mg/m². Dose escalation was planned in cohorts of three to six patients, with 50% increments until grade 1 clinical toxicity (excluding nausea and vomiting) was seen in at least two patients during their first cycle at a given level. Thereafter, 25% dose increments were planned, until dose-limiting toxicity occurred in at least two patients at a given level. Dose-limiting toxicity was defined as a nadir granulocyte count of less than 500/µL at any time, a nadir platelet count of less than 50,000/µL at any time, grade 2 nonhematologic toxicity (excluding nausea, vomiting, and alopecia) occurring before completion of the planned weekly treatments, grade 2 neurotoxicity (excluding grade 2 headache) at any time, grade 3 nonhematologic toxicity occurring after completion of the three weekly treatments, or the need for a treatment delay of more than 2 weeks to permit resolution of toxicity.

The treatment cycle was halted if the platelet count decreased to less than 50,000/µL, the granulocyte count decreased to less than 500/µL, or grade 2 nonhematologic toxicity (excluding nausea, vomiting, and alopecia) occurred. In this case, the cycle was considered complete; treatment could resume 2 weeks later provided the granulocyte count was more than 1,500/µL, the platelet count was more than 75,000/µL, and the patient had recovered from nonhematologic toxicities.

If the preceding cycle was accompanied by minimal clinical toxicity (nadir granulocyte count 1,000/µL, nadir platelet count 75,000/µL, hematologic recovery by day 29, and grade 1 mucositis and diarrhea), the 5-FU dose was increased by 25% in the next cycle, whereas the doses of eniluracil and leucovorin remained the same. The dose of 5-FU was reduced by 33% in the case of a nadir granulocyte count of less than 500/µL, a nadir platelet count of less than 50,000/µL, or grade 3 or 4 nonhematologic toxicity at any time or grade 2 toxicity before completion of the cycle. National Cancer Institute common toxicity criteria, version 1, were used. Fatigue and malaise were considered grade 1 if the symptoms were mild and did not interfere with the patient’s activities; moderate symptoms that affected some specialized activities were scored as grade 2; symptoms were deemed grade 3 if they greatly interfered with the patient’s routine activities and/or were accompanied by a decrease in performance status.

The first three patients were treated weekly for 6 of 8 weeks, but dose-limiting toxicity occurred during the first cycle after 4 or 5 weeks in two patients. The schedule was therefore amended to weekly for 3 of 4 weeks, and a second cohort of patients was treated. Because dose-limiting toxicity occurred in three of six patients in cycle 1, the starting dose was decreased to 10 mg/m². The toxicity associated with the lower dose is described in the Results.

Patient Evaluation and Follow-Up
A blood cell count with WBC differential was obtained weekly. On day 1 of each cycle, liver function tests were performed and blood urea nitrogen, creatinine, and electrolyte levels were determined. Radiographic studies were repeated every third cycle. Treatment was continued until there was evidence of disease progression, provided treatment was tolerated and the patient agreed to continuation of therapy.

Pharmacokinetic Studies
Patients were hospitalized for the 24-hour infusion of 5-FU and after the initial oral dose of 5-FU, to permit extended blood sampling for pharmacokinetic analysis. Blood samples were obtained before treatment, at hours 0.5, 1, 4, 7, 10, 14, 18, 22, and 23 during the infusion, immediately before the end of the infusion, and 60 minutes after the infusion. On the first day of oral 5-FU therapy, blood samples were obtained at the following time points relative to administration of the 5-FU dose: 0.5, 1, 1.5, 2, 3, 6, 9, 12, 15, 18, 22, 23, 24, and 26 hours. The second dose of 5-FU was to be given immediately after the 12-hour blood sample was obtained. The blood was collected into 10-mL heparinized tubes, immediately placed on ice, and centrifuged at 800 x g for 15 minutes; the plasma was then frozen at -70°C and kept at that temperature until the time of analysis. Samples obtained at night were stored on ice and the plasma was isolated the following morning. A 24-hour urine collection was performed on both occasions. The volume of urine was measured at intervals of approximately 8 hours, and an aliquot of urine was transferred to a 50-mL tube and stored on ice. After being transported to the laboratory, the urine was stored at -70°C.

Unless otherwise stated, chemicals were obtained from Sigma Chemical Co (St Louis, MO). 5-FU, uracil, and fluoro-beta-alanine (FBAL) (Fluorochem USA, West Columbia, SC) were measured using validated gas chromatography–mass spectroscopy methods.^30-32 Plasma (100 µL) or urine (10 µL) was spiked with the internal standard, 5-chlorouracil. Pentafluorobenzylbromide was the derivatizing agent. A Supelco (Bellefonte, PA) SPB-20 capillary column was used (15 m x 0.25 mm–inside diameter fused silica, 0.250-µm phase film). Ultrapure helium at a concentration of 1 mL/min was used as the carrier gas. Standard curves were constructed using donor plasma, with 5-FU concentrations ranging from 0.1 to 100 µmol/L, FBAL concentrations from 0.25 µmol/L to 100 µmol/L, and [¹⁵N₂]uracil concentrations from 0.025 to 250 µmol/L.

Noncompartmental pharmacokinetic analytic methods were used to estimate the area under the concentration curve (AUC) using the WinNonLin 2.1 software package (Pharsight, Mountain View, CA). The terminal elimination half-life was estimated from the terminal portion of the concentration-versus-time curve. Oral 5-FU clearance divided by the bioavailability (CL/F) was determined by dividing the administered dose by the AUC extrapolated to infinity. For the IV administration of 5-FU, clearance was determined in the same manner. Peak and trough plasma concentrations were determined by visual inspection of the data.

Measurement of DPD Activity
Venous blood samples were collected in heparinized tubes before treatment, on day 3 of eniluracil therapy (before the morning dose of eniluracil and leucovorin), and on day 8 before eniluracil therapy. The protocol was later amended to permit collection of additional blood samples 12 and 19 days after the last dose of eniluracil whenever possible. Peripheral-blood mononuclear cells were isolated by Ficoll-Hypaque density centrifugation and then subjected to a brief hypotonic lysis (0.2% saline for 30 seconds followed by "rescue" with an equal volume of 1.6% saline) to remove erythrocytes. Intact cell pellets were stored at -70°C until analysis. The frozen cell pellet was suspended in 300 µL of DPD assay buffer (potassium phosphate 35 mmol/L [pH 7.4], magnesium chloride 2.5 mmol/L, and 2-beta-mercaptoethanol 14.3 mmol/L) and placed on ice. The cellular membrane was ruptured by sonication, and the supernatant containing a cellular lysate was collected after centrifugation at 12,000 x g for 30 minutes at 4°C. Protein concentration was determined using the BioRad (Hercules, CA) protein assay kit; bovine serum albumin was used to generate the standard curve. Lysate containing 200 to 300 µg of protein was incubated in a final volume of 1 mL of DPD assay buffer containing [³H]5-FU 20 µmol/L (25 Ci/mmol; Moravek Biochemicals, Brea, CA) and 250 µmol/L nicotinamide adenine dinucleotide phosphate (reduced form) in a 37°C shaking water bath. At 15-minute intervals, a 250-µL aliquot was removed and transferred to a 1.5-mL microfuge tube containing an equal volume of 100% ice-cold methanol. After incubation on ice for at least 15 minutes, the sample was centrifuged, and the methanol-soluble supernatant was filtered through a 0.2-µm Acrodisc filter (Gelman Sciences, Ann Arbor, MI). The samples were then concentrated to dryness in a TurboVap (Zymark Corp, Hopkinton, MA). The samples were resuspended in 200-µL high-performance liquid chromatography (HPLC) mobile phase before analysis.

5-FU was separated from its catabolites by an ion-pairing HPLC method using a Waters (Milford, MA) HPLC system with an in-line Radiomatic 500TR series flow scintillation analyzer (Packard Instruments, Meriden, CT). A 5-µm C18 Columbus Column (4.9 x 250 mm; Phenomenex, Torrey Pines, CA) was used. The mobile phase was tetrabutylammonium hydrogen sulfate 5 mmol/L and KH₂P0₄1.5 mmol/L, pH 9.0, at a flow rate of 1.4 mL/min. Ultima-Flow M (Packard Instruments) at a 3:1 ratio was used for postcolumn scintillation detection. Dihydrofluorouracil and 5-FU eluted at 3 and 13 minutes, respectively. A control sample contained all assay ingredients without a source of protein, to permit correction for any possible degradation of 5-FU or exchange of tritium with water. DPD activity was defined as the amount of catabolites (in picomoles) formed per minute per milligram of protein using the linear portion of the curve.

Statistical and Graphical Analysis
Graphical analysis was performed using SigmaPlot 5.0 for Windows (SPSS, Inc, Chicago, IL), and statistical analysis was performed using SigmaStat for Windows 2.03 (SPSS, Inc). The median time to progression was calculated by a Kaplan-Meier survival curve. The Cockcroft and Gault formula was used as follows to calculate the estimated creatinine clearance: men, (140 - age in years) · (body weight in kg) / (72 · serum creatinine level in mg/dL); women, (0.85) · (140 - age in years) · (body weight in kg) / (72 · serum creatinine level in mg/dL). The strength of linear association between pairs of variables was determined using Pearson’s correlation coefficient: r values .70 reflect a strong correlation, r values between .50 and .70 represent a moderate correlation, and r values of .3 to .5 suggest a weak to moderate correlation. The percent change in nadir granulocyte counts was determined by the following equation: 100 x (baseline value - nadir value) / (baseline value). The relationship between dose and percent change in blood counts was analyzed using a sigmoidal maximum effect model. Coefficients of determination (r²) values greater than .50 indicate a strong fit between the model and the data, whereas values between .25 and .50 signify a moderately strong fit.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patient Characteristics and Clinical Toxicity
Twelve adult patients with good performance status who had undergone a median of two prior chemotherapy regimens participated in this study (Table 1). The majority of patients had adenocarcinomas arising in the gastrointestinal tract. All patients were assessable for toxicity.

A median of 2.5 cycles were given. The worst toxicity experienced by each patient across all cycles of therapy is listed in Table 2. Grade 3 or 4 diarrhea occurred in 33% of patients, and grade 3 fatigue occurred in 25%. Mucositis did not exceed grade 1. Only one patient had a nadir granulocyte count of less than 500/µL. One quarter of patients experienced a nadir hemoglobin level of less than 8.0 mg/dL, but thrombocytopenia was negligible. During the study, one patient with primary bile duct cancer died from refractory upper gastrointestinal bleeding, without thrombocytopenia or coagulation abnormalities. Endoscopy showed no evidence of esophagitis, gastritis, or duodenitis and confirmed that tumor invading the duodenum had caused the bleeding. This death was attributed to rapid progression of the underlying malignancy.

A total of 14 cycles at 15 mg/m² PO bid were given to nine patients (Table 3). Six cycles (43%) were complicated by dose-limiting toxicity, including grade 3 or 4 diarrhea, grade 3 fatigue, and grade 4 granulocytopenia. Three patients received one cycle at 18.75 mg/m². Two patients tolerated this dose but were taken off study because of disease progression. The other patient experienced grade 2 fatigue and requested that the 5-FU dose be reduced to 15 mg/m². A total of 12 cycles at 10 mg/m² PO bid were given to seven patients. Two patients (16.7%) had dose-limiting toxicity (either grade 3 diarrhea or fatigue). No dose-limiting toxicities occurred during 15 cycles at 6.7 mg/m². Therefore, we consider 10 mg/m² PO bid to be a safe starting dosage. In the case of patients treated weekly for 3 of 4 weeks, treatment intervals between cycles were 4 weeks in 49% of cycles and 5 weeks in 30%.

Pharmacokinetic Studies
The plasma concentrations of 5-FU during the initial 24-hour infusion of 2,300 mg/m² are shown in Fig 1. The average 5-FU plasma concentrations 30 minutes and 24 hours into the infusion were 5.8 and 8.0 µmol/L, respectively. One hour after completion of the infusion, the plasma concentration had decreased to 0.16 µmol/L. 5-FU pharmacokinetic parameters are listed in Table 4. Within individual patients, the peak 5-FU plasma concentration was 2.1 ± 0.4-fold (mean ± SD) greater than the trough 5-FU plasma concentration. The average clearance was 1,953 mL/min/m² (median, 1,947 mL/min/m²); for the population, the fastest clearance was 1.9-fold greater than the slowest clearance. Only 1% of the total dose was recovered as parent drug in the urine during the 24-hour infusion.

View larger version (20K): [in this window] [in a new window]   Fig 1. 5-FU plasma levels during the 24-hour infusion of 5-FU 2,300 mg/m2 (top) and after administration of 5-FU 10 to 15 mg/m2 PO bid on day 2 with eniluracil 20 mg PO bid on days 1 through 3 (bottom). Data are presented as mean ± SD.

View larger version (11K): [in this window] [in a new window]   Fig 2. Mean ± SD plasma concentrations of FBAL during the initial 24-hour infusion of 5-FU and 1 hour afterward.

Because the dose of oral 5-FU was rounded down to the nearest 5 mg, the correlation between plasma AUC and 5-FU dose was examined. Over the relatively narrow dose range, the AUC tended to increase with increasing 5-FU dose (Fig 3, top), whereas CL/F was independent of dose (Fig 3, middle). CL/F of 5-FU in the presence of eniluracil correlated closely with creatinine clearance estimated using the serum creatinine level obtained on day 1 of period 2 (Fig 3, bottom). There was no correlation between plasma AUC of 5-FU and severity of diarrhea or absolute nadir granulocyte count (Fig 4).

View larger version (16K): [in this window] [in a new window]   Fig 3. Correlations between 5-FU dose and either AUC0-26 hours (top) or estimated creatinine clearance (middle) and between estimated creatinine clearance and 5-FU clearance (bottom). Pearson’s correlation coefficients are shown.

View larger version (21K): [in this window] [in a new window]   Fig 4. AUC values according to severity of diarrhea (left) or absolute granulocyte count (AGC) nadir (right). Dashed lines represent median values. There was no difference between the AUC values for each group for either diarrhea or AGC nadir.

View larger version (27K): [in this window] [in a new window]   Fig 5. DPD activity in peripheral-blood mononuclear cells during and after eniluracil therapy. Activity (mean ± SD) is expressed as absolute values (top) or percentages of pretreatment values (bottom). The dashed line indicates a putative cut point for DPD deficiency. Numbers of patients are given in parentheses.

In six patients in whom plasma uracil levels were measured 5 or 6 days after the last eniluracil dose, the uracil level had decreased from 45.8 ± 12.2 µmol/L (mean ± SD) on day 3 to 8.5 ± 4.3 µmol/L (20.7% of the peak value). More extended sampling was performed in three patients (Fig 6). The apparent "half-life" of uracil was calculated as 5.5, 3.9, and 4.2 days for the three patients. The measured uracil values had returned to the normal range by day 40 (37 days after the last dose of eniluracil) and day 30 in two of these patients and were predicted to reach the normal range by day 35 in patient no. 6.

View larger version (21K): [in this window] [in a new window]   Fig 6. Uracil plasma levels in three patients during and after eniluracil therapy (20 mg PO bid days 1 to 3). The dashed line represents the upper 99% confidence interval for the pretreatment levels. The regression line is fit between the day 3 uracil value (before the morning dose) and the recovery values.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

A 24-hour continuous infusion of 5-FU 2,300 mg/m² was given on day 2 of period 1 with leucovorin 15 mg PO bid on days 1 through 3 to provide reference pharmacokinetic and pharmacodynamic values. When the pharmacokinetic data for all patients were combined, no clear evidence of diurnal variation in plasma 5-FU levels was found. Within individual patients, the highest plasma 5-FU level was 2.1-fold greater than the lowest plasma 5-FU level. The 5-FU catabolite FBAL accumulated exponentially in the plasma and seemed to reach steady-state by the end of the infusion.

In the presence of eniluracil, FBAL was not detected in plasma during the oral 5-FU dosing period. Although FBAL was detected in the urine, the cumulative amount was approximately 379-fold lower than that measured during the IV infusion of 5-FU. 5-FU CL/F was 33-fold slower in the presence of eniluracil, and the half-life was prolonged to 5.4 hours. Because IV 5-FU was not administered with eniluracil in our study, direct determination of the oral bioavailability of 5-FU was not possible. However, our findings are in close agreement with those of an earlier study by Baker et al,¹⁷ in which the oral bioavailability of 5-FU administered twice daily was essentially 100%. Our estimate (59 mL/min/m²) of CL/F of 5-FU administered with eniluracil is close to that reported in two other studies by Baker et al^17,35 (58 and 51 mL/min/m²). The AUC of 5-FU in our trial did not correlate with the severity of diarrhea. In contrast, Baker et al found that the mean steady-state plasma 5-FU concentration was significantly higher in patients with grade 3 or 4 diarrhea than in those with milder diarrhea, when patients were given eniluracil and 5-FU twice daily for 28 days. The lack of correlation in our trial might be due to the use of a different schedule with higher 5-FU doses, the smaller sample size, or the use of leucovorin. It is also possible that local accumulation of active metabolites of 5-FU in the intestinal mucosa may contribute to gastrointestinal toxicity with this schedule. The AUC did not correlate with either the absolute nadir granulocyte count or the percent change in granulocyte count, but neutropenia was not prominent with this schedule.

Like other investigators,^18,19,29 we also found that DPD activity in peripheral-blood mononuclear cells is profoundly inhibited during eniluracil therapy. We prospectively planned to measure DPD activity in all patients on day 8 of cycle 1 (5 days after the last dose of eniluracil) and found that DPD activity remained markedly depressed in all patients. When this trial was initiated, a 3-week interval between the last dose of eniluracil and subsequent fluoropyrimidine-based therapy was recommended. The rationale behind this recommendation related to the duration of DPD inactivation in a study of oral eniluracil given once daily for 7 days at a dose of 0.74, 3.7, or 18.5 mg/m².¹⁸ In that trial, DPD activity in peripheral-blood mononuclear cells was inactivated within 1 hour of eniluracil dosing and remained inhibited by 93% to 98% 24 hours after dosing.¹⁸ Fourteen days after eniluracil therapy, mean DPD activity was approximately 60% (at the 0.74-mg/m² dose), 70% (3.7-mg/m² dose), and 125% (18.5-mg/m² dose) relative to baseline values. While this trial was in progress, CTEP notified investigators conducting studies of eniluracil therapy that life-threatening or fatal toxicities had been observed in a few patients treated with conventional doses of fluoropyrimidines, despite a 3-week waiting period after the last dose of eniluracil. A 4-week interval was then recommended. Because there was only limited information on the duration of DPD inactivation in clinical studies, and we observed pronounced enzyme inactivation 5 days after completing the first 3 days of eniluracil therapy, we amended our protocol to allow measurement of DPD activity after the completion of a full cycle of eniluracil therapy whenever possible. DPD activity remained substantially inhibited 12 days after eniluracil therapy; 19 days after eniluracil therapy, DPD activity had recovered to 100 pmol/min/mg in only one of five patients. Subsequently, investigators were notified by CTEP that several life-threatening and fatal toxicities had occurred in patients receiving standard-dose fluoropyrimidine therapy 4 and 5 weeks after eniluracil therapy; an 8-week wash-out period is now recommended. The most common schedule for eniluracil and 5-FU in clinical testing is 11.5 and 1.15 mg/m², respectively, PO twice daily for 28 of 35 days.^36,37 Additional studies geared toward characterizing the duration of DPD inactivation are being conducted by the pharmaceutical sponsor. It is critical to determine what level of DPD activity is sufficient to prevent toxicity associated with conventional-dose 5-FU therapy. Although 100 pmol/min/mg has been suggested as a cutoff for severe DPD deficiency, activity levels of 100 to 150 pmol/min/mg have been reported to reflect moderate DPD deficiency.^33,34,38,39

Dramatic increases in plasma and urinary uracil levels are observed in patients with genetically based DPD deficiency, and a similar phenomenon is seen with pharmacologic inactivators of DPD, including eniluracil and sorivudine.^18,29,40-42 The value of monitoring "recovery" of uracil values in place of monitoring recovery of DPD activity is as yet unproven. Plasma uracil levels have generally been measured by HPLC methods. The sensitivity of this approach is hampered by the presence of endogenous uracil in plasma and the limits of ultraviolet absorption detection. Quantitation of uracil by gas chromatography–mass spectroscopy has several advantages. A stable isotope of uracil that contains ¹⁵N rather than ¹⁴N can be used to construct the standard curve, without interference from endogenous uracil. Further, mass spectroscopy has greater sensitivity than does ultraviolet detection. In our trial, baseline uracil levels were detectable in all patients in the submicromolar range (0.09 to 0.25 µmol/L). An unexpected finding was a consistent increase in uracil levels during the initial 5-FU infusion. Although decreased catabolism of uracil by DPD due to competition from 5-FU is a possible explanation, it does not fully account for the observation that the uracil levels continued to increase throughout the 24-hour infusion period. The plasma uracil levels were greatly increased the morning of day 2 of eniluracil therapy and were on average 121-fold greater than pretreatment values. Plasma uracil levels also increased throughout the 24-hour period of oral 5-FU dosing. The average uracil levels between hours 18 and 23 were 2.1-fold higher than the levels observed before the first oral dose of 5-FU, and they were 291-fold higher than pretreatment baseline values. Because DPD is maximally inactivated with bid dosing of eniluracil 20 mg, the additional increase in uracil levels during the oral 5-FU dosing period also suggests that a different process may be involved. Increases in plasma deoxyuridine levels have been reported in patients receiving antifolate thymidylate synthase inhibitors.^42,43 Accumulation of deoxyuridine monophosphate occurs with thymidylate synthase inhibition, and conversion of the monophosphate to deoxyuridine intracellularly leads to efflux of the nucleoside into the extracellular compartment in preclinical models.^44-46 Deoxyuridine can be converted to uracil through the action of thymidine phosphorylase. This phenomenon should be investigated in future studies.

At least two mechanisms may account for the decrease in plasma uracil levels after eniluracil therapy: urinary excretion and recovery of DPD activity. Reversal of the acute 5-FU–associated increase in plasma uracil concentrations may also be a factor. In six patients in whom plasma uracil levels were measured on day 3 of eniluracil therapy and 5 days later, the values had decreased 4.8-fold, consistent with a half-life of approximately 2 days. More extended uracil sampling, performed in three patients, suggested an average half-life of 4.5 days. The projected time to reach the upper 99% confidence limits for pretreatment uracil values averaged 35 days, and the plasma uracil levels were projected to decrease to 1 µmol/L by 24 days. Additional studies are necessary to determine whether patients with normalized plasma uracil concentrations after eniluracil therapy can be safely treated with standard doses of 5-FU. These calculations serve to highlight the potential limitations associated with the use of surrogate markers to assess the pharmacodynamic effect of a drug: depending on the sensitivity of the assay, different conclusions might be drawn concerning when the effect of eniluracil had worn off.

Two schedules of 5-FU–plus–eniluracil therapy have been previously evaluated: one intended to simulate the monthly bolus schedule of 5-FU, and the other intended to simulate a protracted infusion for 4 of 5 weeks. With eniluracil 3.7 mg/m² PO on days 1 through 7, the highest tolerated dose of 5-FU given on days 2 through 6 was 25 mg/m²; the recommended dose of 5-FU in single-agent therapy is 500 mg/m².¹⁹ Thus, eniluracil therapy reduces the tolerated dose of 5-FU 20-fold. When given with 50 mg each of eniluracil and leucovorin on the same schedule, the recommended dose of oral 5-FU was 15 mg/m², which is 28-fold lower than the customary 425/20-mg/m² dose of 5-FU/leucovorin on a monthly schedule.¹⁹ The 28-day schedule involves twice-daily administration of eniluracil and 5-FU, with recommended total daily doses of 11.5 mg/m² (eniluracil) and 1.15 mg/m² (5-FU), given PO for 28 of 35 days. The total daily 5-FU dose is thus 150-fold lower than the customary dose of 300 mg/m² when 5-FU is given as a single agent by protracted infusion. The required 5-FU dose reduction for the schedules involving eniluracil plus 5-FU intended to mimic infusional regimens is greater than the observed reduction in 5-FU clearance. Given that the activity of DPD indirectly influences the amount of 5-FU available for anabolism to its active nucleotide metabolites, a possible explanation is that twice-daily dosing of both 5-FU and eniluracil may result in more sustained exposure of normal tissues to 5-FU, with enhanced intracellular accumulation of cytotoxic 5-FU metabolites. A recent study of the effects of eniluracil on the metabolism of 5-FU in mice bearing colon 38 tumors using ¹⁹F nuclear magnetic resonance spectroscopy confirmed a higher accumulation of 5-FU nucleotides and increased retention of 5-FU in both normal and tumor tissues.⁴⁷

DPD expression may be a predictive factor for response to 5-FU–based therapy.^25-28,48 A recent clinical study has suggested that eniluracil therapy is capable of completely inhibiting DPD activity in tumor tissue.²⁹ It is hoped that pharmacologic inactivation of DPD will provide an opportunity to circumvent a specific mechanism of 5-FU resistance. Preclinical studies have indeed shown an improved therapeutic index with treatment with oral eniluracil plus 5-FU compared with continuous infusion of 5-FU alone.⁴⁹ Ongoing phase III trials comparing treatment with oral 5-FU plus eniluracil and commonly used IV 5-FU regimens will determine whether a similar improvement in clinical outcome can be achieved in cancer patients.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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Submitted March 23, 2000; accepted July 6, 2000.

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