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Phase I and Pharmacologic Study of Oral Fluorouracil on a Chronic Daily Schedule in Combination With the Dihydropyrimidine Dehydrogenase Inactivator Eniluracil

From The Johns Hopkins Oncology Center, Baltimore, MD; University of Alabama, Birmingham, AL; and Glaxo Wellcome, Inc, Research Triangle Park, NC.

Address reprint requests to Sharyn D. Baker, PharmD, Division of Pharmacology and Experimental Therapeutics, The Johns Hopkins Cancer Research Building, 1650 Orleans St, Room 1M87, Baltimore, MD 21207; email sdbaker{at}jhmi.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To determine the maximum-tolerated dose (MTD), toxicities, and pharmacokinetics of oral fluorouracil (5-FU) administered twice daily in combination with oral eniluracil, an inactivator of dihydropyrimidine dehydrogenase, administered for 28 days every 35 days.

PATIENTS AND METHODS: Oral 5-FU 1.35 mg/m² twice daily was administered with oral eniluracil 10 mg daily for 14 to 28 days, followed by a 1-week rest period. Eniluracil was started 1 day before 5-FU. Patients then received escalated doses of oral 5-FU 1.35 to 1.8 mg/m² twice daily with an increased dose of eniluracil 10 mg twice daily for 28 days. A reduced dose of 5-FU 1.0 mg/m² with eniluracil 20 mg twice daily was evaluated.

RESULTS: Thirty-six patients with solid malignancies were enrolled onto the study. Diarrhea was the principal dose-limiting toxicity of oral 5-FU and eniluracil given on this chronic schedule. The recommended phase II dose is 5-FU 1.0 mg/m² twice daily with eniluracil 20 mg twice daily. Mean (SD) values for terminal half-life, apparent volume of distribution, and systemic clearance of 4.5 hours (0.83 hours), 19 L/m² (3.0 L/m²), and 51 mL/min/m² (13 mL/min/m²), respectively. An average of 77% of 5-FU was excreted unchanged in urine after 28 days of treatment. The mean (range) 5-FU C_SS,min values achieved at the 1.0 mg/m² dose level were 22 ng/mL (8 to 38 ng/mL).

CONCLUSION: Chronic oral administration of 5-FU with oral eniluracil is tolerable and produces 5-FU steady-state concentrations similar to those achieved with protracted intravenous administration of 5-FU on clinically relevant dose schedules. Eniluracil provides an attractive means of administering 5-FU on protracted schedules.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
FLUOROURACIL (5-FU) has been the mainstay of therapy for the treatment of metastatic colorectal cancer, despite modest response rates of approximately 20% on conventional bolus administration schedules. Strategies to enhance the antitumor activity of 5-FU have principally focused on increasing conversion of 5-FU to its active anabolites, enhancing binding to thymidylate synthase, and increasing incorporation of its active anabolites into RNA or DNA using diverse approaches, which include biochemical modulation, regional administration, and protracted intravenous (IV) administration.^1,2 Results from a randomized phase II trial demonstrated that protracted IV infusion of 5-FU is as efficacious as and less toxic than 5-FU in combination with leucovorin or L-aspartate,³ and results from a recent meta-analysis suggest that continuous infusion 5-FU may improve the efficacy and safety of 5-FU compared with IV bolus administration.⁴ Although protracted continuous infusion may be more efficacious and less toxic, this method of administration is associated with increased costs, patient inconvenience, and complications related to the requirements for an indwelling IV catheter and for the use of ambulatory infusion pumps.

A different approach to 5-FU modulation involves the catabolic pathway of 5-FU. The first step in 5-FU metabolism is the reduction of 5-FU to 5-fluoro-5,6-dihydrouracil, which has been shown to reduce 5-FU antitumor activity and increase gastrointestinal toxicity in a rat model.⁵ The end product of 5-FU catabolism is alpha-fluoro-beta-alanine (FBAL), which is neurotoxic in animal models and may be responsible for the neurotoxicity observed after 5-FU administration.^6,7 Dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme involved in the catabolism of 5-FU, is found in the liver, intestinal mucosa, pancreas, lung, kidney, and other tissues⁸ and is responsible for the rapid elimination (half-life [t_1/2], 8 to 20 minutes) of more than 70% of an administered dose of 5-FU.⁹ Population studies have demonstrated substantial interpatient variability in DPD activity,^10-13 which most likely accounts for the erratic oral bioavailability (0% to 80%),⁹ wide intersubject variation in 5-FU clearance (> five-fold),^14,15 and high rates of unpredictable toxicity. In addition, tumoral DPD activity has been shown to be a determinant of 5-FU responsiveness in patients with head and neck cancer, among whom those with lower DPD activity are more likely to respond.¹² Therefore, the inactivation of DPD is an attractive target for 5-FU modulation because it may accomplish the following: (1) improve the toxicity profile of 5-FU by decreasing the formation of potentially toxic metabolites, (2) enhance antitumor activity by suppressing 5-FU catabolism in tumor tissue, (3) produce more predictable plasma disposition and toxicologic profiles by decreasing the interpatient variability in systemic clearance, (4) enable predictable oral administration of 5-FU as a result of DPD being responsible for the catabolism and erratic bioavailability of oral 5-FU, and (5) provide the benefits of continuous infusion 5-FU administration without the requirements for central venous access and infusion pumps.

Eniluracil is a mechanism-based, effective inactivator of DPD in vitro¹⁶ and in vivo.¹⁷ In preclinical studies in rats, a single oral dose of eniluracil inactivated more than 99% of hepatic DPD activity,¹⁷ prolonged the t_1/2 of oral 5-FU, and produced 100% oral bioavailability.¹⁸ Recent clinical studies have noted significant changes in the disposition of 5-FU, producing a t_1/2 of 4.5 hours after 5-FU doses of 10 to 25 mg/m²/d given in combination with eniluracil 3.7 mg/m²/d and increased oral bioavailabilty to approximately 100%.¹⁹ Eniluracil doses of 0.74, 3.7, and 18.5 mg/m² inactivated DPD activity in peripheral-blood mononuclear cells within 1 hour of eniluracil administration at all dose levels, which rebounded at 24 hours to approximately 7%, 2.0%, and 2.5% at each dose level, respectively.²⁰ In addition to altering the systemic disposition and increasing the bioavailability of oral 5-FU, eniluracil increased the therapeutic index^18,21 and absolute efficacy²¹ of 5-FU in animal models, most likely by preventing the formation of 5-FU catabolites⁵ and increasing the formation and accumulation of 5-FU anabolites in tumor tissue.^22,23

Eniluracil is being developed to enable chronic oral administration of 5-FU, provide a cost-effective and convenient alternative to protracted IV infusion therapy, and increase the clinical utility and therapeutic index of 5-FU. The principal objectives of this phase I and pharmacologic study were to accomplish the following: (1) determine the maximum-tolerated dose (MTD) of oral 5-FU administered twice daily in combination with oral eniluracil for 28 days every 35 days, (2) describe and quantitate the toxicities of this regimen, (3) characterize the pharmacokinetic behavior of 5-FU and eniluracil after administration of a single dose and after repeated doses, (4) evaluate the effect of varying eniluracil doses on the extent of DPD inactivation, and (5) seek evidence of antitumor activity for this regimen.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patient Selection
Patients with histologically documented advanced solid malignancies refractory to conventional therapy or for whom no effective therapy existed were eligible for this study. Eligibility criteria also included the following: age 18 years; Karnofsky performance status 70% (ambulatory and capable of self-care); life expectancy 12 weeks; adequate hematopoietic (absolute neutrophil count 1,500 neutrophils/mm³, platelet count 100,000 platelets/mm³, hemoglobin 9.0 g/dL), hepatic (total bilirubin 2.0 mg/dL, AST and ALT three times the upper limit of institutional normal [ five times the upper limit of institutional normal if caused by tumor]), and renal (serum creatinine 1.5 mg/dL or creatinine clearance 60 mL/min, estimated according to the method of Cockcroft and Gault²⁴) functions; no history or evidence of malabsorption or processes that might affect gastrointestinal function or surgical resection of the stomach or small bowel; no concurrent treatment with dipyridamole, folinic acid, allopurinol, interferon, metoclopramide, flucytosine, or cimetidine within the previous 2 weeks; no radiation therapy or cytotoxic chemotherapy within 4 weeks of treatment (6 weeks if the previous chemotherapy regimen included mitomycin C or a nitrosourea); no surgery within the previous 2 weeks; and no other coexisting medical conditions of sufficient severity to prevent full compliance with the study. All patients gave informed, written consent according to federal and institutional guidelines.

Dosage and Dose Escalation
Dose of 5-FU. The starting dose of 5-FU, 1.35 mg/m² orally twice daily, was chosen based on the following observations: (1) results from a prior study, 146–001, demonstrated that 5-FU 20 mg/m²/d for 5 days every 28 days in combination with eniluracil 3.7 mg/m²/d was well tolerated and provided a total 28-day exposure of 100 mg/m² and that dividing 100 mg/m² by 28 days yielded a total daily dose of 3.6 mg/m²²⁰; (2) the t_1/2 of 5-FU given with eniluracil was 4.5 hours²⁰; (3) preliminary data from a prior study, 146–002, indicated that the absolute bioavailability of an oral 5-FU solution given with eniluracil was greater than 95%¹⁹; and (4) pharmacokinetic simulations that used parameters calculated for patients in both studies 146–001 and 146–002 demonstrated that an oral 5-FU dose of 1.8 mg/m² twice daily would produce a 5-FU exposure similar to that reported in a study of 5-FU administered as a protracted IV infusion.²⁵ To ensure patient safety, the starting dose of 5-FU was 1.35 mg/m² twice daily for 14 days, which was designated treatment group 1 (Table 1). Regarding the escalation of 5-FU duration, those patients for whom no toxicity was observed in treatment group 1 progressed to treatment group 2, in which they received 5-FU 1.35 mg/m² twice daily for 21 days. In turn, those for whom no toxicity was observed in treatment group 2 were treated with 5-FU 1.35 mg/m² twice daily for 28 days, which was treatment group 3. In this group, two patients mistakenly received 5-FU 1.35 mg/m² twice daily for 28 days with a lower dose of eniluracil 10 mg daily; this dosage level was designated treatment group 3A. Patients in treatment group 3B were treated with 5-FU 1.35 mg/m² twice daily with the higher daily dose of eniluracil 10 mg twice daily for 28 days. After the maximal 5-FU duration was achieved, dose optimization proceeded as follows: doses of oral 5-FU were escalated to 1.8 mg/m² twice daily for 28 days in treatment group 4. In treatment group 5, the dose of 5-FU was decreased to 1.0 mg/m² twice daily because the eniluracil dose was increased to 20 mg twice daily, which could have resulted in increased 5-FU toxicity,²⁰ and dose-limiting toxicity (DLT) was observed in two of six patients treated with 5-FU 1.35 mg/m² twice daily for 28 days during their first course of therapy (treatment group 3B).

Dose of eniluracil. The dose of eniluracil given in treatment groups 1 and 2 was based on initial data from an ongoing phase I study (146–001) in which eniluracil given daily at doses of 3.7 and 18.5 mg/m² seemed to inhibit DPD optimally without drug-related toxicity.²⁰ A fixed dose of 10 mg daily (treatment groups 1 and 2) was used because it provided eniluracil doses within the range of 3.7 to 18.5 mg/m² for patients with body-surface area (BSA) values between 1.1 and 2.7 m² (Table 1). In treatment groups 3 and 4, the eniluracil dose was increased to 10 mg twice daily (20 mg/d) because emerging data from Study 146–001 indicated that DPD activity rebounded to approximately 20%, 5%, and 0% of baseline by 24 hours after treatment with eniluracil doses of 0.74, 3.7, and 18.5 mg/m² daily, respectively.²⁰ For patients with BSA values 2.7 m², a dose of eniluracil 10 mg twice daily (20 mg/d) provided at least 3.7 mg/m²/d but did not exceed 18.5 mg/m²/d, the highest dose level shown to be safe in Study 146–001. In treatment group 3, two patients mistakenly received 5-FU 1.35 mg/m² twice daily for 28 days with a lower dose of eniluracil 10 mg daily, which was designated treatment group 3A. Treatment group 3B consisted of patients being treated with 5-FU 1.35 mg/m² twice daily with a higher daily dose of eniluracil 10 mg twice daily for 28 days. At the time of patient enrollment onto treatment group 5, 5-FU catabolites were measured in urine (during the 24-hour period after treatment) from patients receiving eniluracil doses of 0.74 and 3.7 mg/m²/d in Study 146–001, which suggested incomplete DPD inactivation.²⁰ In contrast, 5-FU catabolites were not detected through 24 hours after treatment in patients who received eniluracil 18.5 mg/m²/d, which indicated 100% DPD inactivation.²⁰ On the basis of these data, the eniluracil dose was increased to 20 mg twice daily (40 mg/day), which provided eniluracil exposures similar to or greater than those achieved at a dose of 18.5 mg/m²/d for patients with BSA values 2.7 mg/m².

Drug Administration
Eniluracil was supplied in the form of 5- and 10-mg white tablets by Glaxo Wellcome, Inc (Research Triangle Park, NC). Eniluracil was administered on days 1 through 15 (for treatment group 1), 1 through 22 (for treatment group 2), and 1 through 29 (for treatment groups 3, 4, and 5). A 1-hour fasting period preceded and followed each dose.

5-FU was supplied in the form of 0.25- and 1-mg white tablets by Glaxo Wellcome, Inc. 5-FU was administered twice daily beginning on day 2 (1 day after the start of eniluracil). The dose of 5-FU was based on BSA, in which the patient’s weight on day 1 of the course was used, and was calculated to the nearest 0.25 mg. The first dose of 5-FU for each day was administered with eniluracil, and the second was administered 12 hours later with the second eniluracil dose (treatment groups 3B, 4, and 5). Patients fasted 1 hour before and after each dose. 5-FU was given twice daily for 14 days (for treatment group 1), 21 days (for treatment group 2), and 28 days (for treatment groups 3, 4, and 5), in each case followed by a 7-day rest period.

Pretreatment and Follow-Up Studies
History assessment, physical examination, routine laboratory studies, and assessment of Karnofsky performance status were performed at baseline and before each course. Ophthalmologic examinations were performed at baseline and after every two courses. Routine laboratory studies, which were obtained weekly, included complete blood cell and differential WBC counts, urinalysis, and evaluation of the levels of electrolytes, creatinine, glucose, albumin, total protein, total bilirubin, alkaline phosphatase, ALT, and AST. Coagulation studies were performed at baseline and weekly thereafter in patients receiving concomitant warfarin or related coumarins. Assessments of measurable or assessable disease were performed at baseline and after every two courses. A complete response was defined as the disappearance of all disease, as documented by two measurements separated by at least 4 weeks, whereas a partial response was defined as at least a 50% reduction in the sum of the products of the bidimensional measurements of all measurable lesions on two assessments separated by at least 4 weeks.

Plasma Sampling and Assay
The plasma disposition of 5-FU, eniluracil, and uracil were evaluated after the morning dose on days 2 and 15 (for treatment group 1), day 22 (for treatment group 2), and day 29 (for treatment groups 3, 4, and 5). Blood samples were collected in heparinized tubes before the administration of the morning dose and at 15 and 30 minutes and 1, 2, 3, 4, 6, 8, and 12 hours after treatment. The 12-hour sample was obtained before the evening dose. Blood samples also were obtained before treatment in the morning on days 2, 8, 15 (except for treatment group 1), 22 (except for treatment groups 1 and 2), and 29. Blood samples were placed immediately in an ice-water bath, and plasma was isolated within 20 minutes of collection using a refrigerated centrifuge and was frozen at -20°C until it was assayed. Urine samples also were collected on days 2 and 29 (except for treatment group 1) after the morning dose to determine concentrations of eniluracil, 5-FU, and FBAL. For treatment groups 2, 3, and 4, urine was collected for 12 hours after treatment. For treatment group 5, urine was collected during the following intervals after treatment: 0 to 6 hours, 6 to 12 hours, and 12 to 24 hours after the evening dose.

For sample analysis, a validated analytic method using derivatization and liquid-liquid extraction with gas chromatography/mass spectrometry detection was used to determine concentrations of 5-FU, eniluracil, uracil, and FBAL in plasma and urine.¹⁹ Whenever possible, samples from the same subject were run in the same analytic batch to minimize the effects of interassay variation. In plasma, the concentration ranges for the calibration curves were 1 to 1,000 ng/mL for eniluracil and 5-FU and 100 to 6,000 ng/mL for uracil. The intra-assay precision (coefficient of variation) and accuracy (bias as percentage of nominal) were as follows: 6.2% to 16.4% and 89.1% to 115.5%, respectively, for eniluracil; 6.8% to 11.4% and 95.7% to 110.5%, respectively, for 5-FU; and 5.1% to 8.8% and 100.8% to 106.9%, respectively, for uracil. In urine, the concentration ranges for the calibration curves were 0.10 to 10 µg/mL, 0.10 to 100 µg/mL, and 10 to 1,000 ng/mL for eniluracil, 5-FU, and FBAL, respectively. For determinations in urine, the intra-assay precision and accuracy were as follows: 5.2% to 8.7% and 99.3% to 102.1%, respectively, for eniluracil; 10.5% to 14.3% and 96.0% to 107.2%, respectively, for 5-FU; and 2.1% to 9.1% and 100.8% to 121.3%, respectively, for FBAL.

Pharmacokinetic Analysis
Individual eniluracil and 5-FU plasma-concentration data that were obtained on day 2 and the final day of drug administration were analyzed by noncompartmental methods²⁷ using the program WinNonlin (Pharsight Corporation, Mountain View, CA). Maximum-plasma concentration (C_max) and time to maximum concentration were determined from the concentration-time curve. The area under the concentration-time curve (AUC) from time 0 to 12 hours (AUC_{0 to 12 hours}) for twice daily dosing and AUC from 0 to 24 hours (AUC_{0 to 24 hours}) for daily dosing was calculated using the linear trapezoidal rule as implemented in WinNonlin. The AUC was extrapolated to infinity (AUC_inf) by dividing the last measured concentration by _z, which was determined from the slope of the terminal phase of the drug concentration-versus-time curve. Oral clearance (Cl/F) was determined by dividing the dose by AUC_inf on the first day of drug administration and by AUC_{0 to 12 hours} or AUC_{0 to 24 hours} on the final day of drug administration. Apparent volume of distribution (V_z/F) was calculated by dividing Cl/F by _z. The accumulation ratios for eniluracil and 5-FU were calculated as the ratio of AUC_{0 to 12 hours} (after multiple doses) and AUC_inf (after a single dose). 5-FU minimum steady-state concentration (C_ss,min) was calculated as the average of the pretreatment concentrations on days 8, 15, 22, and 29. The percentage of eniluracil and 5-FU excreted unchanged in urine was calculated as the amount of total drug measured in the urine collected over 12 hours divided by the administered dose and multiplied by 100. The percentage of 5-FU excreted as FBAL was calculated as the total amount of FBAL measured in urine from 0 to 12 hours after treatment divided by the administered 5-FU dose and multiplied by 100. Uracil C_ss,min was calculated as the average of the pretreatment concentrations on days 8, 15, 22, and 29. Uracil average steady-state concentration (C_ss,ave) was calculated by dividing the AUC_{0 to 12 hours} for the final day of treatment by the dosing interval (tau), which was approximately 12 hours.

Statistical Considerations
A course of treatment was considered complete if 75% of the prescribed study drug was taken. Pharmacokinetic data were described using descriptive statistics. The paired t test was used to compare pharmacokinetic parameters that were determined on the first and last days of 5-FU administration. Univariate correlation analysis was used to assess the relationship between estimated creatinine clearance and drug clearance. Univariate correlation analysis of variance was used to assess the relationship between dose and pharmacokinetic parameters and the relationship between drug exposure (dose, C_max, AUC, and C_ss,min) and National Cancer Institute toxicity grade. If a significant difference was detected by analysis of variance, Tukey’s multiple range test was used to determine which groups differed. The a priori level of significance was set at 0.05. Statistical analysis was performed using the JMP Version 3.1 statistical software program (SAS Institute, Cary, NC).

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Thirty-six patients, whose characteristics are displayed in Table 2, were enrolled onto this study. All patients had received prior chemotherapy. In patients treated with prior 5-FU based regimens, the most prevalent tumor type was colorectal carcinoma. One hundred-two courses were administered, and 96 were completed. Of the six courses that were not completed, treatment was discontinued either because of toxicity (four patients in four courses) or rapid disease progression (two patients in two courses). The median number of courses administered per patient was two (range, one to 10). Patient noncompliance occurred infrequently, with an average of two doses (range, one to three) missed during nine of 96 completed courses. The number of new and total patients and courses, as well as the rates of DLT as a function of 5-FU and eniluracil dose, are listed in Table 1. During the first part of the study, the duration of 5-FU treatment was increased from 14 to 28 days at a dose of 1.35 mg/m² twice daily given in combination with eniluracil 10 mg daily (treatment groups 1 and 2) or 10 mg twice daily (treatment group 3). In treatment group 3, two patients mistakenly received 5-FU 1.35 mg/m² twice daily for 28 days with eniluracil 10 mg daily (treatment group 3A). Three patients were treated with 5-FU 1.35 mg/m² twice daily with an increased daily dose of eniluracil 10 mg twice daily for 28 days (treatment group 3B). No DLT was observed among the first three patients enrolled at this dose level. Therefore, the dose of 5-FU was increased to 1.8 mg/m² twice daily (treatment group 4), which was associated with DLT in two patients during course 1 and in one patient during course 5. The dose of 5-FU was subsequently reduced to the previous dose of 1.35 mg/m² twice daily (treatment group 3B). However, two of three patients (two of six total patients) who were treated at this dose level experienced DLT during course 1, which was an unacceptably high rate of severe toxicity, considering the intention was to develop a 5-FU dose for chronic administration. Therefore, the 5-FU dose was reduced to 1.0 mg/m² twice daily given with eniluracil 20 mg twice daily (treatment group 5), which was associated with DLT in four of 15 patients (one patient during courses 1 to 3, one patient during both courses 2 and 3, one patient during course 3 only, and one patient during course 4 only). At this dose level, one patient completed six courses, two patients completed four courses, and two patients completed three courses of treatment.

Other gastrointestinal toxicities. Nausea and vomiting were observed in patients who were on this chronic oral schedule of 5-FU in combination with eniluracil (Table 1). Nausea and vomiting generally occurred intermittently throughout treatment and responded well to antiemetic medications. Twenty patients (56%) experienced mild-to-moderate nausea and/or vomiting ( grade 2) during 29 of 100 total courses. One patient (one course) who was receiving 5-FU 1.35 mg/m² twice daily with eniluracil 10 mg twice daily developed grade 3 nausea concurrent with grade 2 diarrhea and severe (grade 3) abdominal cramping during course 1. One other patient (two courses) who was receiving 5-FU 1.0 mg/m² in combination with eniluracil 20 mg twice daily experienced dose-limiting grade 4 nausea and vomiting during the second and third courses of therapy. Other observed gastrointestinal effects that were mild-to-moderate in severity included abdominal cramps (nine patients in 11 courses), flatulence (six patients in six courses), and anorexia (five patients in five courses).

Other toxicities. Other nonhematologic toxicities included mucositis, hand-foot syndrome, and fatigue. These toxicities generally were mild-to-moderate and did not seem to be dose-related. Grade 1 mucositis occurred during nine courses involving six patients. Dose-limiting grade 2 mucositis occurred during the fourth course of treatment in one patient who was receiving 5-FU 1.0 mg/m² twice daily and during the first course of therapy in one patient who was treated with 5-FU 1.8 mg/m². In the latter patient, grade 2 mucositis developed concurrently with grade 4 diarrhea. No grade 3 or 4 hand-foot syndrome was observed. Grade 1 or 2 hand-foot syndrome occurred in seven patients (seven courses). Fifteen patients (22 courses) developed grade 1 or 2 fatigue, and one patient at the 5-FU 1.0 mg/m² dose level developed dose-limiting grade 3 fatigue during the third course of treatment, which may have been drug-related.

Mild-to-moderate liver function test abnormalities were noted at all dose levels. Eight patients who received 15 courses developed grade 1 or 2 elevations in total bilirubin level, which generally resolved within 1 week. Four patients (five courses) also developed grade 3 elevations in total bilirubin level, and one patient (one course) experienced a grade 4 elevation; these elevations were concurrent with the progression of hepatic metastases. Grades 1 and 2 elevations in transaminases occurred during 12 courses involving 11 patients, and a grade 3 elevation in transaminases occurred during one course, which may have been related to disease progression. In addition, seven patients (10 courses) experienced grade 1 or 2 elevations in serum creatinine.

One patient experienced delayed neurologic toxicity: a 44-year-old male patient with adenocarcinoma of probable renal origin who received 5-FU 1.0 mg/m² with eniluracil 20 mg twice daily experienced difficulty walking and an unsteady gait. His prior therapy consisted of two chemotherapy regimens that included paclitaxel, ifosfamide, cisplatin, and doxorubicin with the multidrug-resistance reversal agent, VX710. Before study enrollment, he had a mild sensory neuropathy involving the soles of his feet. After 1 year of treatment with 5-FU in combination with eniluracil, he reported difficulty sustaining an erection. Three months later, he developed difficulty walking and unsteadiness of gait. Magnetic resonance imaging that was performed on this patient revealed cerebellar atrophy, particularly in the cerebellar vermis. No metastatic lesions were evident in the brain, and no white-matter changes were evident. Chemotherapy was discontinued, his impotence resolved, and his gait returned to normal over the ensuing 3 months. No new neurologic deficits appeared during the year after the discontinuation of treatment. Magnetic resonance imaging in two other patients who had remained on chronic oral 5-FU and eniluracil treatment for more than 1 year showed no evidence of cerebellar atrophy.

One patient who was receiving warfarin therapy concurrent with 5-FU 1.35 mg/m² twice daily and eniluracil 10 mg daily for 21 days developed an 11-fold increase in the international normalized ratio value relative to baseline on day 30 of course 2, which was concurrent with progressive growth in hepatic metastases. The international normalized ratio values normalized within 7 days after the discontinuation of warfarin and after treatment with transfusions of packed RBCs and fresh frozen plasma.

Hematologic toxicity occurred infrequently in patients on this chronic oral schedule of 5-FU in combination with eniluracil. Two patients (two courses) who were treated with 5-FU 1.0 mg/m² twice daily with eniluracil 20 mg twice daily experienced grade 3 neutropenia. Grade 3 anemia occurred in one patient (one course) who was receiving 5-FU 1.35 mg/m² twice daily in combination with eniluracil for 21 days (treatment phase 2), in which the anemia was concurrent with a drug interaction with warfarin, and in one patient (one course) who was treated with 5-FU 1.0 mg/m² twice daily with eniluracil 20 mg twice daily (treatment phase 5).

Antitumor Responses
Two patients who had developed disease progression during treatment with IV 5-FU–based regimens had a partial response after treatment with 5-FU and eniluracil. A 64-year-old female with adenocarcinoma of the colon with liver metastases had a partial response after four courses of 5-FU 1.8 mg/m² twice daily and eniluracil 10 mg twice daily. The partial response was maintained for 6 months, during which time she received one additional course at the same dosage and five additional courses at the reduced dosage of 5-FU 1.0 mg twice daily and eniluracil 20 mg twice daily. Her prior therapy consisted of four chemotherapy regimens that included 5-FU/levamisole, 5-FU/interferon, 1,3-bis(2-chloroethyl)-1-nitrosourea/vincristine/streptozocin, and the investigational DNA minor groove-binder DMP840. In addition, a 60-year-old male with adenocarcinoma of the colon metastatic to the lungs, who had previously been treated with 5-FU and leucovorin, experienced a partial response after four courses of 5-FU 1.0 mg/m² and eniluracil 20 mg twice daily, which was maintained for 2 months.

Pharmacologic Studies
All 36 patients enrolled onto and treated in this study had complete plasma sampling performed for pharmacokinetic studies on the first day of 5-FU administration (day 2), and 24 patients had complete plasma sampling performed on the final day of 5-FU administration (day 15 for treatment group 1, day 22 for treatment group 2, and day 29 for treatment group 3). Because the extrapolated AUC represented more than 50% of the AUC_inf, 5-FU pharmacokinetic parameters calculated on day 29 from one patient treated in treatment group 3B were excluded from statistical analyses. Mean 5-FU and eniluracil pharmacokinetic parameters are listed in Tables 3 and 4, respectively.

The mean percentage of 5-FU excreted unchanged in urine from time 0 to 12 hours after treatment on day 2 was 43% (range, 12% to 77%); the average percentage excreted at steady-state was 77% (range, 13% to 125%). In the majority of patients, less than 2% of an administered 5-FU dose was excreted in the urine as FBAL. Consistent with the urinary recovery of 5-FU, estimated creatinine clearance correlated well with 5-FU Cl/F on day 2 (R² = 0.466; P < .0001); data from one outlier with a high calculated creatinine-clearance value (341 mL/min) were excluded from this analysis. The relationship between baseline estimated creatinine clearance and 5-FU C_ss,min values is shown in Fig 1, in which a trend toward higher 5-FU C_ss,min values at lower creatinine clearances is evident at the 1.8 mg/m² dose level.

View larger version (14K): [in this window] [in a new window]   Fig 1. Relationship between estimated creatinine clearance at baseline and 5-FU Css,min after chronic oral administration of 5-FU 1.0 mg/m2 twice daily with eniluracil 20 mg twice daily (), 5-FU 1.35 mg/m2 twice daily with eniluracil 10 mg once or twice daily (), and 5-FU 1.8 mg/m2 twice daily with eniluracil 10 mg twice daily (•).

View larger version (11K): [in this window] [in a new window]   Fig 2. Plot depicting the relationship between worst grade of diarrhea during any course and 5-FU Css,min during course 1. The mean 5-FU Css,min value was significantly higher in patients who experienced grade 3 or 4 diarrhea (52 ng/mL) than in those who experienced grade 0 to 2 diarrhea (23 ng/mL; P < .0001).

Eniluracil pharmacokinetics. In general, the pharmacokinetics of oral eniluracil when given in combination were similar to oral 5-FU (Table 4). Peak eniluracil plasma concentrations occurred at median times of 1.1 and 2.0 hours on day 2 and on the last day of eniluracil administration, respectively. Mean eniluracil pharmacokinetic parameters on day 2 included a t_1/2 of 4.0 hours, a V_z/F of 19 L/m², and a Cl/F of 57 mL/min/m². Daily eniluracil doses ranging from 10 to 40 mg did not alter the disposition of eniluracil, and pharmacokinetic parameters were similar on day 2 and after repeated dosing for 15, 22, and 29 days. The mean accumulation ratio was 1.1. No major differences in eniluracil pharmacokinetic parameters were observed at 5-FU doses ranging from 1.0 to 1.85 mg/m² twice daily. The mean percentage of eniluracil excreted unchanged in urine from time 0 to 12 hours after treatment on day 2 was 38% (range, 11% to 67%); the average percentage excreted at steady-state was 46% (range, 34% to 93%).

Inactivation of DPD by eniluracil. Analysis of surrogate markers for the extent of DPD inactivation, which included uracil clearance, 5-FU clearance, and the urinary excretion of 5-FU and FBAL at steady-state, indicated that eniluracil 10 to 20 mg twice daily maximally inactivated DPD. After treatment with eniluracil for 24 hours, uracil concentrations were elevated and reached steady-state between 3 and 8 days at all eniluracil dose levels. After repeated dosing for 15, 22, and 29 days, mean (SD) uracil C_ss,ave values were 3,418 ng/mL (970 ng/mL), 4,368 ng/mL (1,483 ng/mL), and 3,868 ng/mL (791 ng/mL) at the 10 mg daily, 10 mg twice daily, and 20 mg twice daily eniluracil dose levels, respectively; mean (SD) values for uracil C_ss,min were 3,571 ng/mL (1,262 ng/mL), 3,963 ng/mL (1,287 ng/mL), and 3,760 ng/mL (817 ng/mL), respectively. 5-FU Cl/F values were similar in each eniluracil dosing group (Table 3), and the percentage recovery of unchanged 5-FU in urine during the 12-hour period after treatment was independent of eniluracil dose, with median values of 92%, 72%, and 79% at the 10 mg daily, 10 mg twice daily, and 20 mg twice daily eniluracil dose levels, respectively. Urinary excretion of FBAL was less than 2% of an administered 5-FU dose in all but one patient, who received an eniluracil dose of 10 mg daily (8%).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The inactivation of DPD by eniluracil represents an important strategy to increase the clinical utility and therapeutic index of 5-FU, which historically has been associated with unpredictable pharmacologic behavior and toxicity. Variation in tumoral DPD activity has been correlated with response to 5-FU in patients with head and neck cancer.¹² In preclinical studies, the oral DPD inactivator eniluracil increased the efficacy and the therapeutic index of 5-FU in several animal tumor models,^18,21 possibly by suppressing the catabolism and increasing the formation and accumulation of 5-FU anabolites in tumor tissue.^22,23 In addition to potentially circumventing 5-FU resistance mechanisms, eniluracil altered the pharmacologic profile of 5-FU. The inactivation of DPD by eniluracil increased the oral bioavailability of 5-FU to approximately 100%¹⁹ and reduced systemic clearance 20-fold to values comparable to the glomerular filtration rate (50 to 60 mL/min/m²).^19,20 Eniluracil also enabled the oral administration of 5-FU on a 5-day dosing schedule without adversely altering the toxicologic profile of 5-FU.^19,20 This clinical study demonstrated that a chronic oral regimen of 5-FU administered in combination with eniluracil resulted in a tolerable and predictable toxicity profile. The 5-FU plasma concentrations that were achieved were comparable to those obtained during protracted IV 5-FU infusions.^25,28 In the presence of eniluracil, the majority of 5-FU was excreted unchanged in the urine. Renal excretion, instead of DPD-related catabolism, is the major route of elimination of 5-FU when given in combination with eniluracil. By making all patients virtually DPD deficient, eniluracil may improve the clinical, pharmacologic, and safety profile of 5-FU when it is administered orally at reduced doses and as dosing guidelines based on renal function are formulated. Chronic oral administration of 5-FU is feasible and may provide the benefits of continuous infusion 5-FU administration without the requirements for central venous access and infusion pumps.

The toxicities reported in this study were similar to those that were noted to occur after 5-FU was administered as a continuous IV infusion at a dose of 300 mg/m²/d on days 1 through 28 every 35 days³ and, in general, were similar to those observed for protracted continuous IV infusion of 5-FU alone.²⁸ Intolerable diarrhea precluded the chronic administration of oral 5-FU of doses more than 1.0 mg/m² twice daily in combination with eniluracil 20 mg twice daily when given for 28 days every 35 days. To determine whether the occurrence of diarrhea was attributed, in part, to 5-FU pharmacokinetics, relevant parameters of 5-FU exposure were correlated with the severity of diarrhea. The mean 5-FU C_ss,min value was significantly higher in patients who experienced grade 3 or 4 diarrhea than in patients who experienced less severe diarrhea (52 v 23 ng/mL, respectively; P < .0001). Because the high 5-FU concentrations observed during course 1 for some patients were not always observed in the same cycle of treatment in which grade 3 or 4 diarrhea occurred (courses 1 to 6), elevated 5-FU plasma concentrations may only be a marker for patients who are at risk for developing severe diarrhea. Similar associations have been observed between 5-FU steady-state plasma concentrations and toxicity after protracted infusion of 5-FU alone.^29,30 5-FU AUC values on day 2 of course 1 also were higher in patients who experienced grade 3 or 4 diarrhea than in those who did not, during any course (702 v 413 ng/mL/hr, P < .0001). 5-FU AUC values were highly correlated with C_ss,min values (R² = 0.690; P < .0001), and it is unknown which parameter is best for predicting toxicity. Indeed, the causal relationship between elevated 5-FU AUC or C_ss,min values and the occurrence of severe diarrhea during all courses of treatment remains to be demonstrated prospectively. Such relationships could be exploited to prospectively reduce 5-FU doses early in the course of treatment to avoid the onset of severe toxicity later in the course of therapy. Unlike with continuous IV infusion schedules of 5-FU, hand-foot syndrome occurred infrequently after chronic oral administration of 5-FU with eniluracil. Given that this schedule of oral 5-FU achieves plasma concentrations similar to those obtained with continuous IV schedules and given the small amount of DPD activity observed in patients treated with eniluracil, the uncommon occurrence of hand-foot syndrome may be a result of the absence of circulating 5-FU catabolites.

Severe neurologic toxicity developed in one patient after the patient received treatment with 5-FU and eniluracil for 1 year. 5-FU given alone may produce acute neurotoxicity.²⁸ Preclinical data have implicated the 5-FU metabolite FBAL in 5-FU–induced neurotoxicity.^6,7 In addition, a neurologic syndrome characterized by cerebellar ataxia or a slowly reversible encephalopathic syndrome has been observed in patients who have severe DPD deficiency after receiving 5-FU.^31-34 The involvement of FBAL or other 5-FU metabolites is unlikely in patients with severe DPD deficiency who do not generate FBAL via the 5-FU catabolic pathway. Instead, these neurotoxic reactions have been correlated with prolonged exposure to elevated plasma levels of 5-FU in DPD-deficient patients.^31,33 Given the small amount of DPD generated in patients treated with eniluracil, 5-FU seems to be the cause of delayed neurotoxicity in this patient.

The systemic disposition of oral 5-FU administered chronically at doses ranging from 1.0 to 1.8 mg/m² twice daily in combination with eniluracil was nearly identical to that observed in a previous study of oral 5-FU 10 to 25 mg/m² daily for 5 days with eniluracil,¹⁹ which indicates dose-independent 5-FU pharmacokinetics over a broad range of 5-FU doses. In the presence of eniluracil, strong correlations between 5-FU clearance and calculated creatinine have been noted previously.¹⁹ In the present study, the amount of 5-FU excreted in urine at steady-state was determined, and results showed that the majority of an administered 5-FU dose was eliminated as unchanged drug (mean, 77%). The principal route of 5-FU elimination has been shifted from DPD-related metabolism to renal excretion. At the recommended phase II dose of 5-FU 1.0 mg/m² twice daily in combination with eniluracil, the mean (range) 5-FU C_ss,min achieved was 22 ng/mL (8 to 38 ng/mL), which is equivalent to that that occurs after 5-FU 300 mg/m²/d administered as a protracted infusion (range, 2 to 38 ng/mL).²⁵ The pharmacokinetics of oral eniluracil were similar to that of 5-FU (Tables 3 and 4), and the accumulation of both eniluracil and 5-FU after repeated dosing for 28 days was negligible. Oral 5-FU and eniluracil can be given chronically in combination with predictable exposure to these drugs.

Several surrogate markers were used to determine the minimum effective dose of eniluracil for optimal DPD inactivation. Because uracil is an endogenous substrate for DPD³⁵ and because more than 95% inactivation of DPD by eniluracil seems to be required to maintain maximally elevated plasma uracil concentrations in vivo,³⁶ plasma uracil concentrations were used as an indicator of the extent of DPD inactivation. In individuals with normal DPD activity, plasma uracil concentrations are maintained at levels below 100 ng/mL.³⁴ In this study, uracil steady-state concentrations averaged from 3,000 to 4,000 ng/mL and seemed to reach slightly higher maximum levels in patients who received eniluracil 10 to 20 mg twice daily. 5-FU systemic clearance and renal excretion also were used to assess DPD inactivation; both parameters were independent of eniluracil dose. The urinary recovery of FBAL, the final product of 5-FU catabolism, was the final surrogate marker for the extent of DPD inactivation. When 5-FU is given alone, more than 50% of the 5-FU dose is recovered in the urine as FBAL,⁹ and it is expected that a higher extent of DPD inactivation would correspond with a lower recovery of urinary FBAL. In this study, a negligible fraction of 5-FU was recovered in the urine as FBAL (< 2.0%) in most patients. Results from another study demonstrated that a higher daily eniluracil dose of 50 mg/d did not further reduce the recovery of 5-FU as FBAL in urine.²⁰ The slightly higher percentage of FBAL (8%) excreted in one patient who received eniluracil 10 mg daily with 5-FU 1.35 mg/m² twice daily suggests that doses of 10 to 20 mg twice daily may provide more optimal inactivation of DPD. Combined, these data indicate that eniluracil doses of 10 to 20 mg twice daily may be considered to maximally inactivate DPD.

Although 28 of the 36 patients in this study had been treated previously with 5-FU–based therapy, partial responses were observed in two patients with metastatic adenocarcinoma of the colon who had previously developed progressive disease while receiving IV 5-FU–based therapy. On the basis of these results, a phase II trial of 5-FU 1.0 mg/m² given with eniluracil 10 mg twice daily for 28 days every 35 days was initiated in patients with previously untreated metastatic colorectal cancer.³⁷ Several pivotal trials in progress are evaluating the relative merits of an oral schedule versus a protracted IV schedule of 5-FU or IV 5-FU plus leucovorin in patients with metastatic colorectal cancer.

In summary, the results of this study indicate that chronic oral administration of 5-FU with eniluracil is a tolerable and active treatment regimen. Oral 5-FU 1.0 mg/m² twice daily in combination with eniluracil 20 mg twice daily produces steady-state concentrations similar to those achieved after protracted IV infusions at clinically relevant dose schedules. In addition, eniluracil does not adversely alter the toxicologic profile of 5-FU when given orally on a 28-day chronic schedule repeated every 35 days. Indeed, eniluracil provides an attractive means of administering 5-FU on protracted schedules. The use of oral 5-FU with eniluracil allows the circumvention of the many disadvantages of protracted IV administration, which are associated with increased costs, greater patient inconvenience, and catheter-related infections and vascular complications. However, it will be important to define the overall impact of oral 5-FU/eniluracil dosing schedules on the therapeutic index of 5-FU in the next phase of developmental studies.

	ACKNOWLEDGMENTS

 
Supported by a grant from Glaxo Wellcome, Inc, Research Triangle Park, NC, and by grant no. RR-00035 to the Adult General Clinical Research Center, The Johns Hopkins Hospital, Baltimore, MD.

We thank Mary Duerr for assistance with study coordination and data collection, Yelena Zabelina for assistance with sample processing, and the referring medical staff and medical and nursing staffs of The Johns Hopkins Oncology Center, Baltimore, MD, and the University of Alabama, Birmingham, AL, for the care of the patients in this study.

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Submitted June 3, 1999; accepted October 8, 1999.

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