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Eniluracil Treatment Completely Inactivates Dihydropyrimidine Dehydrogenase in Colorectal Tumors

From the Departments of Medicine and Therapeutics and Pathology, Institute of Medical Sciences, University of Aberdeen, and Department of Surgery, Aberdeen Royal Infirmary, Aberdeen; and Department of Oncology, Glaxo Wellcome, Greenford, United Kingdom; and Laboratory of Genetic Metabolic Diseases, Emma Childrens Hospital, Amsterdam Medical Center, Amsterdam, the Netherlands.

Address reprint requests to Howard L McLeod, MD, Department of Medicine and Therapeutics, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, United Kingdom; email h.l.mcleod{at}abdn.ac.uk

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To determine the effect of eniluracil on colorectal tumor dihydropyrimidine dehydrogenase (DPD) activity.

PATIENTS AND METHODS: Patients who were to undergo primary colorectal tumor resection received oral eniluracil 10 mg/m² twice daily for 3 days before surgery. Mononuclear cells were obtained before the start of eniluracil and on the morning of surgery, to measure DPD activity, protein, and mRNA. Plasma uracil was also measured at these two time points to assess the effect of eniluracil on pyrimidine accumulation. DPD activity, protein, and mRNA were also assessed in colorectal tumors and adjacent normal mucosa of patients who received eniluracil and untreated control patients.

RESULTS: DPD activity in tumors from 10 untreated patients ranged from 30 to 92 pmol/min/mg of protein. In contrast, there was no detectable tumor DPD activity in 10 patients who received eniluracil. A similar pattern was observed in mononuclear cells, where median pretherapy activity was 366.5 pmol/min/mg of protein (range, 265 to 494 pmol/min/mg of protein) and was undetectable immediately before surgery. Plasma uracil changed from a median less than 0.2 µmol/L before therapy to 27.76 µmol/L before surgery. No difference in DPD protein or mRNA was observed between pretherapy and presurgery mononuclear cell samples or between treated and untreated tumor samples.

CONCLUSION: This study provides definitive evidence that eniluracil completely inactivates DPD activity in human solid tumors. The increased plasma uracil and decreased DPD activity are consistent with systemic inactivation of the enzyme. The mechanism of inactivation is at the catalytic level, because no changes in DPD protein or mRNA were observed. Treatment with eniluracil will eliminate DPD activity as a source of pharmacokinetic fluorouracil variability or resistance in human colorectal cancer.

	INTRODUCTION

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WITH NEARLY 40 YEARS of clinical use, fluorouracil (5-FU) is the third most commonly prescribed anticancer agent.¹ 5-FU has single-agent activity against colorectal cancer and is part of combination chemotherapy for breast, head/neck, and upper gastrointestinal tumors.^2,3 Given the heavy usage of this chemotherapy agent, strategies to improve 5-FU antitumor activity and development of more convenient administration approaches are constantly under investigation.^3,4

The majority (~85%) of an administered 5-FU dose is metabolized by dihydropyrimidine dehydrogenase (DPD) to inactive metabolites.⁵ Oral bioavailability of 5-FU is highly variable and unpredictable (0% to 80%).⁶ Gastrointestinal and hepatic DPD activity is thought to be responsible for the highly variable bioavailability of oral 5-FU, which limits the agent to intravenous routes of administration. This is especially troublesome in the context of prolonged continuous infusion therapy, where the added antitumor activity has to be balanced with the increased risk of complications associated with this route of administration.^4,7 DPD is also a potential source of resistance to 5-FU. The ratio of DPD activity in tumor versus adjacent normal tissue was significantly higher in patients with head/neck cancer who did not achieve an objective response to 5-FU–based chemotherapy than in patients who did objectively respond to this therapy.⁸ This is consistent with 5-FU resistance because of increased inactivation by DPD. Intratumoral DPD activity has also been observed in colorectal and hepatocellular carcinomas.^9,10

Eniluracil (5-ethynyluracil,776C85; Glaxo Wellcome, Research Triangle Park, NC) is an uracil analog with an ethynyl group at the 5' position.¹¹ Although it closely resembles 5-FU, eniluracil has no direct antitumor activity at conventional doses.^11-13 However, it is a potent inactivator of DPD.^11,14 Preclinical studies of eniluracil have shown increased 5-FU plasma half-life and area under the concentration-time curve.¹¹ In animal models, the therapeutic index and efficacy of 5-FU seems to be enhanced with the addition of eniluracil, possibly by the prevention of the formation of 5-FU catabolites, which are toxic and have no antitumor activity.^12,13 Preclinical data suggest that 5-FU catabolites may actually inhibit or interfere with antitumor effects.¹² Therefore, eniluracil is being developed to enable the oral administration of 5-FU and potentially modulate 5-FU cytotoxicity.

The impact of DPD inactivation on 5-FU has been demonstrated in the phase I studies. The recommended phase II dose of 5-FU, when combined with eniluracil, was 1 to 1.15 mg/m² bid for 28 days or 25 mg/m² daily for 5 days every 4 weeks, as compared with 300 mg/m²/d and 500 to 1,000 mg/m² with 5-FU alone.^15,16 These early clinical studies have demonstrated complete inhibition of mononuclear-cell DPD activity in patients who received either a single dose or multiple doses of eniluracil.^15,16 In addition, indirect evidence for systemic DPD inhibition has been provided by the observed increase in plasma uracil in patients who received eniluracil. Such changes in plasma uracil would only be expected if more than 90% of DPD was inactivated.^16,17

However, no data are available to demonstrate DPD inactivation in human tumor tissue. The objective of this study was to evaluate the ability of eniluracil to inactivate DPD activity in the target tissue, colorectal tumor, after the preoperative oral administration of eniluracil to patients who required surgical resection of a primary colorectal cancer.

	PATIENTS AND METHODS

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Patient Eligibility
Patients were required to have a radiologically or histologically confirmed diagnosis of colorectal cancer that would require primary surgical resection. Patients had to be more than 18 years of age, able to swallow and retain oral medication, and have adequate hematologic, renal, and hepatic function. Patients were excluded if they had been treated with 5-FU prodrugs or other fluoropyrimidines within 1 week before enrollment, or if they had a malabsorption syndrome or significant gastrointestinal dysfunction, had a concurrent uncontrolled medical condition, received treatment with previous preoperative radiotherapy to the primary lesion, or had any psychologic condition that precluded informed consent. Patients who received warfarin or interferon were excluded. Written informed consent was obtained from all patients before enrollment onto the study. The study was approved by the Grampian Research Ethics Committee.

Pretreatment Assessment
Before initiation of therapy, all patients had a physical examination and a medical history was obtained. Laboratory tests for hematology and clinical biochemistry were assessed and weight and height determined for calculation of body surface area.

Study Design
The study hypothesis was that DPD activity in eniluracil-treated tumors will be completely inactivated. Therefore, the inclusion of at least 10 patients in both the eniluracil and untreated group would give 80% power to detect a significant difference at the P < .05 level. A total of 14 patients received eniluracil 10 mg/m² orally twice daily for 3 days before surgery (Table 1). The final dose was taken on the morning of surgery, between 4 and 10 hours before surgery (mean, 6 hours). Blood samples were obtained, before the start of the eniluracil administration and on the morning of surgery, for measurement of mononuclear-cell DPD activity, protein, and mRNA and plasma uracil. Tumor and adjacent normal tissue was also obtained from 10 eniluracil-treated patients and from 10 consecutive, noneniluracil-treated control patients who were to undergo resection for colorectal cancer.

Tissue Preparation
Peripheral-blood mononuclear cells (PMNCs) were isolated from 20 mL of blood by density centrifugation through Ficoll-Hypaque and washed in phosphate-buffered saline before storage in NaPO₄ buffer 35mmol/L with 10% glycerol.⁹ Plasma was obtained from 10 mL of heparinized blood. Both PMNCs and plasma were stored at -80°C until required.

Colonic resection specimens were rapidly transported from the operating room to the pathology department. Viable tumor and normal mucosa from at least 10 cm away from the tumor were dissected immediately after excision by an experienced pathologist (G.I.M.) and snap frozen in liquid nitrogen. The same pathologist carried out a histologic review to determine Dukes' stage and differentiation status and to assess the degree of tumor cellularity in the samples. All tumors were adenocarcinomas of varied Dukes' stage (Table 1).

Cytosol Preparation
PMNCs were lysed in 300 µL of buffer A (KPO₄ buffer 35 mmol/L, pH 7.4, MgCl₂ 2.5 mmol/L, 2-mercaptoethanol 10 mmol/L) by three cycles of freeze/thawing on dry ice.⁹ Cell debris was pelleted by centrifugation at 12,000 x g for 20 minutes at 4°C, and the cytosolic fraction was removed and stored at -80°C until required. Tissue was homogenized in 2 mL of buffer A with 0.25 mol of sucrose, benzamidine 1 mmol/L, aminoethylisouronium bromide 1 mmol/L, and EDTA 5 mmol/L.⁹ The homogenate was centrifuged at 100,000 x g for 60 minutes at 4°C. The cytosolic fraction was stored at -80°C until required.

DPD Activity Measurement
DPD activity was determined using a previously described method.⁹ In brief, cytosol (50 µL) was incubated in a reaction mixture that contained reduced nicotinamide adenine dinucleotide phosphate 125 µmol/L, [¹⁴C]-5-FU 20 µmol/L, KPO₄ buffer 35 mmol/L, pH 7.4, MgCl₂ 2.5 mmol/L, and 2-mercaptoethanol 10 mmol/L. The mixture had a final volume of 125 µL and was incubated at 37°C for 60 minutes. The reaction was terminated by the addition of an equal volume of ice-cold ethanol. The supernatant was assayed for 5-FU catabolites using high-performance liquid chromatography with on-line radioactivity detection. Each sample was analyzed in triplicate, and a negative control (no reduced nicotinamide adenine dinucleotide phosphate) was included for each sample. A positive control (human liver cytosol) was included with each assay run. DPD activity was expressed as pmol of catabolite formed per minute and was normalized for cytosolic protein concentration (pmol/min/mg protein). The limit of quantitation of this assay is 1 pmol/min/mg protein. The intra- and interassay coefficients of variation for DPD activity from human liver cytosol were less than 8%.

Immunoblot Analysis
A rabbit polyclonal antibody was generated against human DPD using a peptide for amino acids 1,006 to 1,020. This antibody was specific for a 105 kd band in cytosol from human liver, colon, and PMNCs on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Fifty micrograms of cytosolic protein from tissue lysates were resolved on a 7.5% polyacrylamide gel.¹⁸ The resolved proteins were transferred to a nitrocellulose membrane and blocked with 5% concentrated low-fat milk in Tris-buffered saline, 0.1% Tween (TBST) for 2 hours at room temperature, before incubation with the primary antibody (1/2,000 dilution in 5% milk TBST) for 2 hours at room temperature. The membrane was then incubated with a horseradish peroxidase–linked secondary antibody, followed by detection by chemiluminescence. Intensity of DPD protein expression was then determined using densitometry.

Quantitation of DPD mRNA by Reverse Transcriptase-Competitive Polymerase Chain Reaction
DPD mRNA levels were quantitated in paired samples of pretreatment and presurgery PMNCs or colorectal tumor and normal mucosa tissue by a previously described method (Johnston et al, manuscript submitted for publication). First-strand cDNA was synthesized from 10 µg of total RNA. A competitive polymerase chain reaction (PCR) assay was then performed against 1 µL of cDNA. PCR products were resolved on 2.5% agarose gel and visualized by ethidium bromide staining. Gel images were scanned and PCR products evaluated using Molecular Analyst software (BioRad, Hercules, CA) to quantify sample mRNA content.

Analysis of Plasma Uracil
Plasma uracil concentration was determined using high-performance liquid chromatography by a previously described method.¹⁹ The limit of quantitation of this assay was 0.2 µmol/L.

Statistical Analysis
Comparison of DPD activity, protein, mRNA, and plasma uracil concentrations in paired pretreatment and presurgery samples was made using the Wilcoxon test. Comparison of DPD activity, protein, and mRNA between control and eniluracil-treated colorectal tissues was performed using the Mann-Whitney test.

	RESULTS

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Inactivation of DPD in Colorectal Tumor
DPD activity was measured in matched colorectal tumors and normal mucosae in 10 of 14 patients who received eniluracil and 10 untreated patients. Of the four unassessable samples, the tumor of one patient was not resectable, the resection sample from two tumors contained only enough tissue for histopathology purposes, and one was inadvertently placed in formalin fixative. DPD activity was below the limit of detection in all normal mucosa and tumor samples from eniluracil-treated patients. In contrast, DPD activity was measurable in all untreated samples (median activity: normal mucosa, 70 pmol/min/mg protein [range, 40 to 105]; tumor, 64.5 pmol/min/mg protein [range, 30 to 92]; Fig 1).

View larger version (9K): [in this window] [in a new window]   Fig 1. DPD activity in colorectal tumor from 10 untreated patients and 10 patients receiving eniluracil 10 mg/m2 orally twice daily for 3 days before surgery.

Evidence for Inactivation of DPD by Eniluracil Using Surrogate Markers
The influence of eniluracil on systemic DPD was evaluated in 14 patients with blood samples taken before administration of eniluracil and before surgery. PMNC DPD activity was detected in all 14 pretreatment samples (median activity, 366.5 pmol/min/mg protein; Table 2). All patients exhibited PMNC DPD activity in the expected range, and a high degree of interpatient variability was observed (range, 265 to 494 pmol/min/mg protein; Table 2). Complete inactivation of PMNC DPD was observed (< 1 pmol/min/mg protein) in all presurgery samples (Fig 2A). A significant increase in uracil concentration was also observed after eniluracil treatment (median plasma uracil concentration pretreatment, 0.2 µmol/L; before surgery, 27.76 µmol/L; P < .001; Fig 2B). This is consistent with systemic inactivation of DPD because uracil is a natural substrate for DPD.

View larger version (17K): [in this window] [in a new window]   Fig 2. Influence of eniluracil treatment on (A) PMNC DPD activity or (B) plasma uracil.

Mechanism of Interaction
Immunoblotting for DPD protein was performed in five paired PMNC samples (Fig 3). DPD protein was not significantly different between paired pretreatment to presurgery samples (mean ratio, 1.27; P = .34). No significant difference between pretreatment and presurgery PMNC DPD mRNA levels was observed (mean ratio, 1.07; P = .6), which indicates that there is no evidence for a compensatory increase in DPD mRNA after inactivation by eniluracil. DPD protein was also present in three eniluracil-treated and three control tissue samples, and no significant difference in protein level was determined between eniluracil-treated or untreated samples for either normal mucosa or tumor tissue (P = .83).

View larger version (31K): [in this window] [in a new window]   Fig 3. Paired PMNC samples from five patients are shown, demonstrating no change in DPD protein after treatment. Also shown are the DPD activity and mRNA measurements for the same PMNC samples, which show alteration in catalytic activity but not mRNA expression after eniluracil treatment.

Side Effects
Eniluracil therapy was well tolerated by all 14 patients. One patient reported occasional mild dizziness and headache during the 3 days of eniluracil treatment. No other side effects were reported.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
This study provides definitive evidence that eniluracil completely inactivates tumor DPD activity. DPD activity in colorectal tumors from control patients ranged from 30 to 90 pmol/min/mg protein, similar to that previously described in 60 randomly selected patients.⁹ In contrast, there was no detectable DPD activity in any of the tumor samples from patients who received eniluracil therapy. The enzyme inactivation was not specific to tumor tissue because there was no detectable activity in PMNCs or normal bowel mucosae from eniluracil-treated patients (Table 2). This is consistent with a previous report in which no detectable PMNC DPD activity was observed 1 hour after eniluracil 18.5 mg/m².¹⁶ In addition, plasma uracil values increased from undetectable levels to 27.76 µmol/L, consistent with systemic inactivation of DPD activity. Preclinical studies demonstrated that an increase in plasma uracil is only detectable after more than 90% inactivation of DPD activity has occurred.¹⁷ An eniluracil dosage of less than 10 mg/m² taken orally twice daily, as used in this study, may also completely inactivate intratumoral DPD. Because there is a low incidence of eniluracil-associated side effects, dosage alterations are not warranted. However, the formulation used in clinical trials is a combination tablet with a fixed amount of eniluracil and 5-FU. Therefore, reductions of 5-FU dosage, because of toxicity, will also decrease the eniluracil dosage.

Although in vitro studies have indicated that eniluracil acts by covalent binding to a cysteinyl residue in the DPD protein, which prevents substrate binding and subsequent catalysis, there was previously no data on the in vivo mechanism of this interaction.¹⁴ Eniluracil-induced alterations at the protein or mRNA level could explain alterations in DPD activity. However, through the use of immunoblot analysis, no difference in DPD protein levels was observed between pretreatment or presurgery PMNC samples. In addition, DPD protein levels were not significantly different in tumors from patients treated with eniluracil when compared with untreated controls. A similar picture was observed for DPD mRNA, where eniluracil treatment did not produce a consistent change in DPD expression. Together these data demonstrate an interaction at the catalytic level, with no change in DPD protein. This has important clinical implications because it illustrates that the synthesis of new DPD protein will be the rate-limiting step in the regeneration of systemic DPD activity. There is currently an empirical recommendation that 5-FU not be administered at conventional doses until at least 8 weeks after cessation of eniluracil therapy. In the rat, DPD protein has a half-life of 60 hours, which suggests that it would take approximately 15 days (six half-lives) after discontinuing eniluracil to reach steady-state levels of active enzyme.¹⁷ The kinetics of DPD regeneration in humans are not yet known, thus preventing more definitive recommendations for the minimum waiting period required before conventional-dose 5-FU administration after eniluracil therapy. However, it is unlikely that a patient will need to reach steady-state levels of DPD protein to adequately metabolize conventional doses of 5-FU, because only patients with significant reductions in DPD activity are at risk for severe 5-FU toxicity.^20,21

Complete inactivation of DPD activity by eniluracil has significant implications for the use of fluoropyrimidine chemotherapy. Early clinical studies have already demonstrated that coadministration of eniluracil leads to complete oral bioavailability of 5-FU.¹⁵ Oral therapy with eniluracil and 5-FU seems to be well tolerated in multiple-dose studies.^15,16 Although interpatient variation in 5-FU bioavailability has been observed (range, 72% to 207%),¹⁵ much of this variability may be because of interpatient variation in renal function, which becomes the predominant route of 5-FU elimination after eniluracil treatment.¹⁵ The influence of eniluracil on intrapatient variability in 5-FU pharmacokinetics has not been published. Our results are consistent with systemic inactivation of DPD, removing DPD as a barrier to intracellular 5-FU absorption, distribution, or retention.

Variability in 5-FU pharmacokinetics has important clinical implications. Previous studies have demonstrated a relationship between systemic exposure and patient survival.²² In addition, previous studies in head/neck cancer have suggested the presence of a subset of patients with up to 6.7-fold higher tumor DPD than that observed in adjacent normal tissue.⁸ The ratio of DPD activity in tumor to normal tissue was significantly higher in patients without an objective response to 5-FU–based chemotherapy, consistent with inactivation by DPD before the formation of cytotoxic nucleotides. These data are consistent with in vitro studies that demonstrate an influence of high DPD activity on 5-FU cytotoxicity.²³ However, a clear role for DPD in regulating 5-FU activity in colorectal tumors has not been directly evaluated. In this study, treatment with eniluracil completely inactivated tumor DPD activity in all patients, eliminating DPD activity as a source of 5-FU resistance in patients with colorectal cancer.

A previous study observed higher DPD activity in normal bowel tissue than tumor tissue, which suggests that differences in 5-FU degradation may contribute to the relative therapeutic index of 5-FU.⁹ Because complete inactivation of DPD activity occurred in both tumor and adjacent normal tissues from patients receiving eniluracil, DPD-directed therapy will not directly influence the therapeutic index of 5-FU between normal and malignant tissue. However, eniluracil is not a substrate for the enzymes involved in 5-FU activation to cytotoxic nucleotides.¹¹ Because enzymes such as thymidine phosphorylase, thymidine kinase, and uridine phosphorylase are known to be overexpressed in tumor versus normal tissue, eniluracil treatment should not influence the relative tumor-specific activity of 5-FU.^24,25 In addition, preclinical data suggest that DPD inactivation may improve the therapeutic index of 5-FU by decreasing the production of 5-FU catabolites. Administration of dihydrofluorouracil resulted in toxicity but no antitumor activity in preclinical models.¹² Potential benefits of DPD inactivation have also been suggested from early clinical studies, eg, a lower than expected incidence of hand-foot syndrome has been observed with a 28-day regimen of oral 5-FU and eniluracil.²⁶ The results of current phase III studies will help guide the place of eniluracil plus 5-FU in the treatment of colorectal cancer.

	ACKNOWLEDGMENTS

 
Supported by an investigator-driven research grant from Glaxo Wellcome, United Kingdom.

We thank Thomas Spector for advice, discussion, and encouragement during the course of this study.

	NOTES

 
F.Y.H. and S.J.J. contributed equally to this work.

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Submitted January 15, 1999; accepted April 6, 1999.

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