Cancer Chemotherapy & Biotherapy: Principles & Practices, 4th Edition

5-Fluoropyrimidines

Jean L. Grem

The 5-fluorinated pyrimidines were rationally synthesized by Heidelberger et al.1 on the basis of the observation that rat hepatomas use radiolabeled uracil more avidly than nonmalignant tissues, a finding that suggested differences in the enzymatic pathways used for uracil metabolism. Fluorouracil (5-FU) has become particularly useful in the treatment of gastrointestinal (GI) adenocarcinomas and squamous cell carcinomas arising in the head and neck. Synergistic interaction of 5-FU and related fluoropyrimidines with other antitumor agents, irradiation, and physiologic nucleosides, and enhanced activity of 5-FU activity by leucovorin (LV), have also invoked interest.

STRUCTURE AND CELLULAR PHARMACOLOGY

The chemical structures of the initial two 5-fluoropyrimidines to enter clinical trials are shown in Figure 7-1. The simplest derivative, 5-FU (molecular weight [MW]= 130), has the slightly bulkier fluorine atom substituted at the carbon-5 position of the pyrimidine ring in place of hydrogen. The key features of 5-FU are outlined in Table 7-1. Activation to the nucleotide level is essential to antitumor activity. The ribonucleoside derivative 5-fluorouridine (FUrd) has been used exclusively in preclinical studies. The deoxyribonucleoside derivative 5-fluoro-2′-deoxyuridine (FdUrd, MW= 246) is commercially available (floxuridine, FUDR) and is primarily used for hepatic arterial administration.

Transport

5-FU shares the same facilitated-transport system as uracil, adenine, and hypoxanthine. In human erythrocytes, 5-FU and uracil exhibited similar saturable (K [(binding affinity] ~4 mmol/L; Vmax 500 pmol/sec per 5 µL cells) and nonsaturable (rate constant approximately 80 pmol/sec per 5 µL cells) components of influx. The system is neither temperature-dependent nor energy-dependent.2, 3 5-FU permeation is pH-dependent. Ionization of the hydroxyl group attached to the fourth carbon (pK [ionization constant of acid]= 8.0) markedly depresses its transmembrane passage. 5-FU entry into erythrocytes via nonfacilitated diffusion and a facilitated nucleobase transport system clearly differs from that used by pyrimidine nucleosides.3 In rat hepatoma cells, maximal accumulation of free intracellular 5-FU occurs within 200 seconds2; total intracellular 5-FU increases thereafter as the result of formation of nucleotides and RNA incorporation.

FdUrd is a nucleoside. There are at least four major nucleoside transport (NT) systems in mammalian cells that vary in substrate specificity, sodium dependance, and sensitivity to nitrobenzylthioinosine.4 Two basic classes of human NT systems are present: equilibrative (bidirectional) and concentrative (sodium-dependent, unidirectional). Human equilibrative NT (ENT-1) and concentrative NT (CNT-1) are selective for pyrimidines; the former is present in most cell types, including cancer cells, and the latter is present in liver, kidney, intestine, choroid plexus, and some some tumor cells. In Ehrlich ascites cells, intracellular FdUrd reaches equilibrium with extracellular drug within 15 seconds.5 Total intracellular drug continues to accumulate thereafter from rate-limiting phos-phosphorylation to form fluorodeoxyuridylate (5-fluoro-2′-deoxyuridine-5′monophosphate, FdUMP) and other nucleotides.

Metabolic Activation

Activation of 5-FU to the ribonucleotide level may occur through one of two pathways, as outlined in Figure 7-2 6, 7, 8, 9, 10, 11, 12, 13, 14, 15: direct transfer of a ribose phosphate to 5-FU from 5-phosphoribosyl-1-pyrophosphate (PRPP) as catalyzed by orotic acid phosphoribosyl transferase (OPRTase); the addition of a ribose moiety by uridine (Urd) phosphorylase followed by phosphorylation by Urd kinase. Sequential action of uridine/cytidine monophosphate kinase (UMP/CMP kinase) and pyrimidine diphosphate kinase result in the formation of fluorouridine diphosphate (FUDP) and fluorouridine triphosphate (FUTP); the latter is incorporated into RNA by the action of RNA polymerase.

Figure 7.1 Structures of pyrimidine ring, 5-fluorouracil, and 5-fluoro-2′-deoxyuridine.

The pathway catalyzed by OPRTase may be of primary importance for 5-FU activation in healthy tissues because its inhibition by a nucleotide metabolite of allopurinol diminishes toxicity to bone marrow and GI mucosa,7, 12, 14 but it is also the dominant route of 5-FU activation in many murine leukemias.6 Other cancer cell lines appear to activate the drug by the action of Urd phosphorylase and Urd kinase.7, 8, 9, 10, 11, 12, 13, 14 Although one activation pathway may appear to predominate in a given cancer cell under certain conditions, both pathways can often be used.

In the presence of a 2′-deoxyribose-1-phosphate (dR-1-P) donor, 5-FU is converted to FdUrd by thymidine (dThd) phosphorylase.15, 16 dThd kinase then forms FdUMP, a potent inhibitor of thymidylate synthase (TS). FdUMP can be also be formed by ribonucleotide reductase-mediated conversion of FUDP to fluorodeoxyuridine diphosphate (FdUDP), followed by dephosphorylation to FdUMP. FdUMP and FdUDP are substrates for thymidine monophosphate and diphosphate kinases, respectively, resulting in the formation of fluorodeoxyuridine triphosphate (FdUTP). FdUTP can be incorporated into DNA by DNA polymerase.

Physiologic Urd metabolites are largely present in vivo as nucleotide sugars that are necessary for the glycosylation of proteins and lipids, which plays an important role in cytoplasmic and cell membrane metabolism. 5-FU nucleotide sugars, such as FUDP-glucose, FUDP-hexose, FUDP-N-acetylglucosamine, and FdUDP-N-acetylglucosamine, have been detected in mammalian cells.17, 18, 19 The extent to which 5-FU nucleotide sugars are incorporated into proteins and lipids and any possible metabolic consequences are unclear.

Catabolic enzymes also play important roles in nucleoside metabolism. Acid and alkaline phosphatases nonspecifically remove phosphate groups to convert nucleotides to nucleosides. 5′-Nucleotidases also remove a phosphate group from the nucleotide. Nucleosidases break the glycosyl linkage to release a free base. Pyrophosphatases remove two phosphate groups from the 5′-position of the nucleotide, with release of a monophosphate. The pyrimidine phosphorylases catalyze the reversible conversion of pyrimidine base to nucleoside. FdUrd serves as a substrate for both Urd and dThd phosphorylases in a tissue-dependent manner, yielding 5-FU.15, 16 dThd phosphorylase is homologous to platelet-derived endothelial growth factor, which is involved in angiogenesis.

MECHANISM OF ACTION

Inhibition of Thymidylate Synthase

At least two primary mechanisms of action appear capable of causing cell injury: inhibition of TS and incorporation into RNA. FdUMP binds tightly to TS and prevents formation of thymidylate (thymidine 5′-monophosphate, dTMP), the essential precursor of thymidine 5′-triphosphate (dTTP), which is required for DNA synthesis and repair. The functional TS enzyme comprises a dimer of two identical subunits, each of MW ~30 kd (bacterial) or ~36 kd (human). Each subunit has a nucleotide-binding site and two distinct folate binding sites, one for 5,10-methylenetetrahydrofolate (5,10-CH FH4) monoglutamate or polyglutamate, and one for dihydrofolatepolyglutamates. FdUMP competes with the natural substrate 2′-deoxyuridine monophosphate (dUMP) for the TS catalytic site.20, 21 During methylation of dUMP, transfer of the folate methyl group to dUMP occurs by elimination of hydrogen attached to the pyrimidine carbon-5 position (Fig. 7-3). This elimination cannot occur with the more tightly bound fluorine atom of FdUMP, and the enzyme is trapped in a slowly reversible ternary complex (Fig. 7-4). The “thymineless state” that ensues is toxic to actively dividing cells. Toxicity can be circumvented by salvage of dThd in cells that contain dThd kinase. The circulating concentrations of dThd in humans are not thought to be sufficient (approximately 0.1 µmol/L) to afford protection.22 The plasma levels of dThd are approximately 10-fold higher in rodents, which complicates preclinical evaluation of the antitumor activity of various TS inhibitors.

The reduced-folate cofactor is required for tight binding of the inhibitor to TS. The natural cofactor for the TS reaction, 5,10-CH FH, in its monoglutamate and polyglutamate forms, binds through its methylene group to the carbon-5 position of FdUMP. The polyglutamates of 5,10-CH FH4 are much more effective in stabilizing the ternary complex.23, 24 Most other naturally occurring folates also promote FdUMP binding to the enzyme, but form a more readily dissociable complex. Polyglutamated forms of dihydrofolic acid (FH) promote extremely tight binding of FdUMP to the enzyme.25, 26 FH accumulates in cells exposed to methotrexate (MTX). Although MTX is a relatively weak inhibitor of TS in cell-free experiments, MTX polyglutamates are more potent inhibitors.26 MTX polyglutamates decrease the rate of ternary complex formation among FdUMP, folate cofactor, and TS. The ability of MTX polyglutamates to inhibit ternary-complex formation is influenced by the glutamation state of the reduced-folate cofactor and is substantially reduced in the presence of 5,10-CH FH4 pentaglutamate.26 Similarly, in tissue culture, MTX-induced depletion of intracellular reduced folates causes a marked reduction in the rate of formation of ternary complex.25, 27

TABLE 7.1 KEY FEATURES OF 5-FLUOROURACIL

Mechanism of action:

Incorporation of fluorouridine triphosphate into RNA interferes with RNA synthesis and function.
Inhibition of thymidylate synthase by fluorodeoxyuridylate (FdUMP) leads to depletion of thymidine 5′ monophosphate and thymidine 5′ triphosphate, and accumulation of deoxyuridine monophosphate and deoxyuridine triphosphate.
Incorporation of fluorodeoxyuridine triphosphate and deoxyuridine triphosphate into DNA may affect DNA stability. Genotoxic stress triggers programmed cell death pathways.

Metabolism:

Converted enzymatically to active nucleotide forms intracellularly.
DPD catalyzes the initial, rate-limiting step in 5-fluorouracil (5-FU) catabolism.

Pharmacokinetics:

Primary half-life is 8–14 minutes after IV bolus.
Nonlinear pharmacokinetics from saturable catabolism: Total-body clearance decreases with increasing doses; clearance is faster with infusional schedules.
Volume of distribution slightly exceeds extracellular fluid space.

Elimination:

Approximately 90% is eliminated by metabolism (catabolism → anabolism).
<3% and < 10% unchanged drug excreted by kidneys with infusional and bolus 5-FU.
Reduction of 5-FU to dihydrofluorouracil by DPD is rate-limiting. Thereafter: dihydrofluorouracil →fluoroureidopropionic acid → fluoro-β-alanine.
5-FU and its catabolites undergo biliary excretion.

Pharmacokinetic drug

Interference with 5-FU catabolism markedly prolongs its half-life.

   Interactions:

Inhibitors of DPD:
   Thymidine and thymine
   Uracil (component of uracil and ftorafur)
   5-chloro-2,4-dihydroxypyridine (component of ftorafur, 5-chloro-2,4-dihydroxypyridine, and potassium oxonate)
   3-cyano-2,6-dihydroxypyridine (component of emitefur, 3-{3-[6-benzoyloxy-3-cyano-2-pyridyloxycarbonyl]benzoyl}-1-ethoxymethyl-5-fluorouracil)
   (E)-5(2-bromovinyl)uracil (metabolite of sorivudine)
   Eniluracil
   Chronic administration of cimetidine (but not ranitidine) may decrease the clearance of 5-FU
   Dipyridamole increases 5-FU clearance during continuous i.v. infusion.
   Interferon-α may decrease 5-FU clearance in a dose- and schedule-dependent manner.

Biochemical drug interactions

Thymidine salvage via thymidine kinase repletes thymidine 5′ triphosphate pools, decreases FdUMP formation, and antagonizes the DNA-directed toxicity of 5-FU and 5-fluoro-2′deoxyuridine; thymidine may increase fluorouridine triphosphate formation and its incorporation into RNA.
Sequential methotrexate 5-FU increases 5-FU toxicity and increases fluorouridine triphosphate (FUTP) incorporation into RNA; may antagonize DNA-directed toxicity of 5-FU.
Leucovorin increases intracellular pools of reduced folates; 5,10-methylenetetrahydrofolate polyglutamates enhance the stability of reduced folate-FdUMP–thymidylate synthase ternary complex; the magnitude and duration of thymidylate synthase inhibition is increased.
Inhibitors of de novo pyrimidine synthesis (N-phosphonoacetyl-l-aspartic acid, brequinar) increase 5-FU anabolism to the ribonucleotide level and 5-FU–RNA incorporation; uridine triphosphate, cytidine triphosphate, deoxycytidine triphosphate, and deoxyuridine monophosphate depletion may enhance RNA- and DNA-directed toxicity of 5-FU

Toxicity:

Gastrointestinal epithelial ulceration
Myelosuppression
Dermatologic
Ocular
Neurotoxicity (cognitive dysfunction and cerebellar ataxia)
Cardiac (coronary spasm)
Biliary sclerosis (hepatic arterial infusion of FdUrd)

Precautions:

Nonlinear pharmacokinetics: difficulty in predicting plasma concentrations and toxicity at high doses.
Patients with deficiency of DPD may have life-threatening or fatal toxicity if treated with 5-fluoropyrimidines.
Duration of DPD inhibition with eniluracil may be prolonged (8-week washout period recommended).
Patients receiving sorivudine should not receive concurrent 5-fluoropyrimidines (4-week washout period recommended).
Older, female, and poor-performance–status patients have greater risk of toxicity.
Closely monitor prothrombin time and INR in patients receiving concurrent warfarin DPD, dihydropyrimidine dehydrogenase.

Figure 7.2 Intracellular activation of 5-fluorouracil (5-FU). dUTP, deoxyuridine triphosphate; FdUDP, fluorodeoxyuridine diphosphate; FdUMP, fluorodeoxyuridylate; FdUrd, 5-fluoro-2′-deoxyuridine; FdUTP, fluorodeoxyuridine triphosphate; FUDP, fluorouridine diphosphate; FUMP, fluorouridine monophosphate; FUrd, 5-fluorouridine; FUTP, fluorouridine triphosphate; PPRP, phosphoribosyl phosphate.

The kinetics of formation and dissociation of the ternary complex have been studied using bacterial enzyme.27 After binding of inhibitor and 5,10-CH FH4 to the first catalytic site in the free enzyme, the second site becomes exposed. The two binding sites seem to be nonequivalent in terms of their dissociation constants, with Kd values of 1.1 × 10e-11 and 2 × 10e-10 mol/L.29 FdUMP binds less avidly to the mammalian enzyme, with a dissociation half-life (t1/2) of 6.2 hours.30

Elucidation of the crystal structure of TS has permitted a complex kinetic and thermodynamic description of ternary complex formation.31, 32 The interaction proceeds by an ordered mechanism with initial nucleotide binding followed by 5,10-CH FH4 binding to form a rapidly reversible noncovalent ternary complex. Enzyme-catalyzed conversions result in the formation of a covalent bond between carbon-5 of FdUMP and the one-carbon unit of the cofactor.
The overall dissociation constant of 5,10-CH FH4 from the covalent complex is approximately 1×10e-11 mol/L.

Figure 7.3 Synthesis of thymidylate from deoxyuridylate

The three-dimensional conformation of free and bound TS has been characterized through molecular modeling and iterative crystallographic analysis of bacterial enzymes.31, 32, 33 Although the MW and amino acid composition of the bacterial and mammalian enzymes differ, the primary sequence and the active site residues show high homology.34 Thus, the bacterial ternary complex has served as a surrogate for the design of novel inhibitors of human TS.

Figure 7.4 Interaction of fluorodeoxyuridylate (FdUMP) with thymidylate synthase.

Despite the high specificity and potency of TS inhibition by FdUMP and the well-established lethality of dTMP and dTTP depletion, inhibition of TS is not the sole cause of 5-FU toxicity. If 5-FU toxicity results from dTTP depletion, then dThd should reverse the toxic effects. Examples of complete protection from 5-FU cytotoxicity by dThd have been reported, but dThd shows variable effectiveness in rescuing cells exposed to 5-FU.35, 36 Murine lymphoma cells experience an early phase of toxicity during the initial 24 hours of 5-FU exposure, associated with S-phase accumulation.35 Addition of dThd prevents the S-phase block induced by dTTP depletion and abolishes early growth inhibition. After 24 hours (approximately one cell-cycle length) of 5-FU incubation, dThd no longer prevents lethality, and toxicity is maximal for cells exposed during G1 phase of the cell cycle; this phenomenon is attributed to progressive incorporation of 5-FU into RNA. In another model, a 3-hour incubation with 5-FU at 5 to 20 µmol/L produces a dThd-reversible toxicity, although dThd could not reverse toxicity associated with high 5-FU concentrations.36

Experimental evidence from in vivo studies supports the concept that 5-FU toxicity is at least partially independent of its effect on TS. Coadministration of 5-FU and dThd prevents the early inhibition of DNA synthesis, but markedly increases 5-FU toxicity to healthy tissues in the whole animal, increases the antitumor effect of 5-FU against various animal tumors, and increases [3H]FUrd incorporation into RNA.37, 38, 39 Other pharmacologic measures that increase FUTP formation and its RNA incorporation also increase its toxicity.37, 40

RNA-Directed Effects

The contribution of RNA-directed toxicity to ultimate lethality varies greatly, depending on the type of cancer cell and the experimental conditions. 5-FU is extensively incorporated into nuclear and cytoplasmic RNA fractions, which may result in alterations in RNA processing and function, such as inhibiting the processing of initial pre-rRNA transcripts to the cytoplasmic rRNA species in a dose- and time-dependent manner (Table 7-2).40, 41, 42, 43, 44, 45, 46

Net RNA synthesis may be inhibited during and after fluoropyrimidine exposure in a concentration- and time-dependent fashion. In some cancer cell lines, a highly significant relationship exists between 5-FU incorporation into total cellular RNA and the loss of clonogenic survival.8, 47, 48 5-FU is incorporated into all species of RNA; substantial amounts of [3H]5-FU accumulate in low-MW (4S) RNA at lethal drug concentrations.45 Although the analog replaces only a small percentage of uracil residues in RNA, the incorporated 5-FU residues appear to be stable and to persist in RNA for many days after drug administration.36, 50,51

5-FU exposure affects mRNA processing and translation. Polyadenylation of mRNA is inhibited at relatively low concentrations of 5-FU,52, 53 and altered metabolism of specific proteins such as dihydrofolate reductase (DHFR) precursor mRNA has been reported.54 Incorporation of 5-FU into RNA may affect quantitative and qualitative aspects of protein synthesis.55, 56, 57, 58, 59

In vitro-transcribed TS mRNA with 100% substitution of 5-FU leads to alteration in the secondary structure of mRNA, but no differences in the translational efficiency.58 In another system, 100% substitution of uracil residues in human-TS complementary DNA (cDNA) with either FUTP or 5-bromouridine 5′-triphosphate (BrUTP) indicated that the translational rate is inhibited only in the presence of BrUTP-substituted cDNA.59 The stability of the transcribed mRNA in a cell-free system is increased by threefold and 10-fold with FUTP and BrUTP, respectively, and nondenaturing gel electrophoresis shows different conformations for each of the substituted mRNA species.

TABLE 7.2 RNA-DIRECTED CYTOTOXIC EFFECTS OF 5-FLUOROURACIL (5-FU)

Decrease in net RNA synthesis.
Inhibition with RNA processing.
Inhibition of messenger RNA polyadenylation.
Alteration of the secondary structure of RNA.
5-FU residues in transfer RNA form covalent complex with enzymes involved in posttranslational modification of uracil residues.
Incorporation into uracil-rich small nuclear RNA species interferes with normal splicing.
Quantitative changes in protein synthesis.
Qualitative changes in protein synthesis.
Up-regulation of thymidylate synthase (TS) protein synthesis.
TS bound in ternary complex cannot bind to TS messenger RNA.
TS protein translation no longer repressed.

Changes in the structure and levels of small nuclear RNAs (snRNA) and small nuclear ribonuclear proteins (snRNP) result from 5-FU treatment.60, 61, 62, 63, 64,65 With 8% replacement with 5-FU in HeLA cells, the levels of U2-snRNA and U2-snRNPs decrease in nuclear extracts.62 The substitution of FUTP for uridine triphosphate (UTP) in a cell-free system (84% replacement of uracil residues by 5-FU) leads to pH-dependent missplicing of [32P]-labeled human β-globin precursor mRNA; pH values favoring 5-FU ionization promote missplicing.63

The splicing reaction of precursor RNA exposed to either 1 mmol/L UTP or FUTP in Tetrahymena rRNA, an autocatalytic, self-splicing system with one intron and two exons, reveals that the rate and extent of formation of all RNA product species is decreased with 100% FUTP substitution.64 Further, 5-FU substitution greatly increases the pH and temperature sensitivity of the process. Partial ionization of 5-FU residues at physiologic pH (pK 5-FU= 7.8 versus pK uracil= 10.1) may therefore destabilize the active conformation of RNA.65

Another potential locus of 5-FU action is inhibition of enzymes involved in posttranscriptional modification of RNA.43, 66, 67, 68, 69 5-FU exposure inhibits tRNA uracil 5-methyltransferase, which may result in decreased formation of modified Urd (pseudouridine) bases in tRNA.66 In the presence of S-adenosylmethionine, 5-FU-substituted tRNA forms a stable covalent complex containing uracil 5-methyltransferase, 5-FU-tRNA, and the methyl group of S-adenosylmethionine, suggesting that irreversible inhibition of RNA methylation contributes to RNA-directed cytotoxicity.67 5-FU-substituted yeast glycine tRNAs form highly stable covalent complexes with pseudouridine synthase, which might interfere with the posttranscriptional modifications of many nucleotide positions in tRNA, rRNA, and snRNA that are otherwise converted to pseudouridine. 5-FU incorporation alters the biosynthesis of U2-snRNA with subsequent effects on snRNA-protein interactions.69 Even low levels of 5-FU incorporation (5% replacement) inhibit the formation of pseudouridine.69 Subtle changes in the structures of these essential splicing cofactors may thus have profound effects on snRNP- precursor mRNA interactions and interfere with precursor mRNA splicing.

Although 5-FU-associated cytotoxicity in cancer cells exposed in the presence of sufficient concentrations of dThd to circumvent TS inhibition is presumed to result from RNA-directed effects of 5-FU, it is paradoxical that significant incorporation of 5-FU into RNA may occur in some cancer cell lines in the absence of toxicity. The factors that influence whether 5-FU–RNA incorporation results in cytotoxicity are not clear. The rate of RNA incorporation and the species into which the fluoropyrimidine is incorporated may be more important determinants of cytotoxicity than the total amount incorporated. 5-FU and FUrd may be channeled into different ribonucleotide compartments and, ultimately, into distinct classes of RNA.70

In summary, the changes that result in altered pre-RNA processing and mRNA metabolism are not uniform for all RNA species after 5-FU exposure. Effects on precursor and mature rRNA, precursor and mature mRNA, tRNA, and snRNA species suggest inhibition of processing; incorporated 5-FU residues also inhibit enzymes involved in posttranscriptional modification of uracil. Many of the RNA-directed effects of 5-FU undoubtedly occur as a consequence of its fraudulent incorporation into various RNA species. However, rapid changes in mRNA levels suggest that at least some of these alterations may be mediated by other 5-FU-associated alterations in cellular metabolism or posttranslational modification.71 The changes in certain key mRNAs resulting from 5-FU exposure may be relevant as a mechanism of cytotoxicity. 5-FU-mediated interference with the production of enzymes involved in DNA repair may have cytotoxic consequences, such as 5-FU-mediated inhibition of ERCC-1 mRNA expression in cisplatin-resistant cancer cells.72 5-FU and FUrd produce structural and functional alterations in uracil-rich snRNAs, and consequently in snRNPs with potential repercussions on cellular growth and metabolism. The RNA-directed effects of 5-FU are even more complex than previously appreciated, and some RNA effects may be independent of 5-FU incorporation into RNA.

DNA-Directed Cytotoxic Mechanisms

The biochemical consequences of TS inhibition and the potential effects on DNA integrity are summarized in Table 7-3. Inhibition of TS results in depletion of dTMP and dTTP, thus leading to inhibition of DNA synthesis and interference with DNA repair. Accumulation of dUMP occurs behind the blockade of TS, and further metabolism to the deoxyuridine triphosphate (dUTP) level may occur.73, 74, 75 Inhibition of TS is accompanied by elevated concentrations of deoxyuridine in the extracellular media in cell culture models and in plasma of rodents; monitoring changes in plasma deoxyuridine levels may, therefore, serve as an indirect reflection of TS inhibition.

TABLE 7.3 DNA-DIRECTED CYTOTOXIC EFFECTS OF 5-FLUOROURACIL (5-FU)

Biochemical consequences of thymidylate synthase inhibition
Deoxyribonucleotide imbalance
Depletion of thymidine monophosphate and thymidine triphosphate
Accumulation of deoxyuridine monophosphate
Elevation of extracellular deoxyuridine
Formation of deoxyuridine triphosphate
Accumulation of deoxyadenosine triphosphate
Direct and indirect effects on DNA synthesis and integrity
Inhibition of net DNA synthesis
“Uracil” misincorporation into DNA (fluoro- and deoxyuridine triphosphate)
Interference with nascent DNA chain elongation
Altered stability of nascent DNA
Induction of single-strand breaks in nascent DNA
Interference with DNA repair
Induction of single- and double-strand breaks in parental DNA
Induction of programmed cell death

FdUTP and dUTP are substrates for DNA polymerase, and their incorporation into DNA is a possible mechanism of cytotoxicity.76, 77, 78, 79, 80, 81, 82, 83, 84, 85 5-FU cytotoxicity in some models correlates with the level of 5-FU-DNA.80, 81, 84 Two mechanisms prevent incorporation of FdUTP and dUTP into DNA. The enzyme dUTP pyrophosphatase or dUTP hydrolase catalyses the hydrolysis of FdUTP to FdUMP and inorganic pyrophosphate.86, 87 The DNA repair enzyme uracil-DNA-glycosylase hydrolyzes the fluorouracil-deoxyribose glycosyl bond of the FdUMP residues in DNA, thereby creating an apyrimidinic site.82, 84, 88 The bare deoxyribose 5′-monophosphate is subsequently removed from the DNA backbone by an AP (apurinic/apyrimidinic) endonuclease, creating a single strand break, which is subsequently repaired. With thymidine triphosphate depletion, however, the efficiency of the repair process is substantially weakened. Uracil-DNA-glycosylase is a cell cycle-dependent enzyme with maximal levels of activity at the G1 and S interface, such that excision of the fraudulent bases occurs before DNA replication. The activity of uracil-DNA-glycosylase inversely correlates with the level of FdUrd incorporation into DNA in human lymphoblastic cells.80 Because the affinity of human uracil-DNA-glycosylase is much lower for 5-FU than for uracil, it is removed more slowly from DNA by this mechanism.88Recent studies suggest that FdUTP inhibits the activity of uracil-DNA-glycosylase.89 Accumulation of deoxyadenosine triphosphate (dATP) accompanies TS inhibition.90, 91, 92 The combined effects of deoxyribonucleotide imbalance (high dATP, low dTTP, high dUTP) and misincorporation of FdUTP into DNA may have several deleterious consequences affecting DNA synthesis, the integrity of nascent DNA, and induction of apoptosis.

A variety of DNA-directed effects have been described.93, 94, 95, 96, 97, 98, 99, 100, 101 5-FU treatment inhibits DNA elongation and decreases the average DNA chain length.79, 93, 94 DNA strand breaks accumulate in 5-FU-treated cells and correlate with excision of [3H]5-FU from DNA.93 5-FU and FdUrd result in single- and double-stranded DNA breaks in HCT-8 cells in a concentration- and time-dependent fashion, a process that is enhanced by LV and limited by dThd.95, 96FdUrd exposure may result in the formation of large (one to five-megabase) DNA fragments as a result of double-strand DNA breaks; the time course and extent of DNA megabase fragmentation correlates with loss of clonogenicity in HT29 cells.97 The pattern of DNA fragmentation is distinct from that associated with gamma radiation, which produces random breaks. The pattern of high-MW DNA damage differs in SW620 cells, which are equally sensitive as HT29 cells to FdUrd-induced inhibition of TS, but require higher drug concentrations and longer exposures to achieve a comparable degree of DNA fragmentation and cytotoxicity. The basis is higher activity of dUTPase and failure to accumulate dUTP.98 Simple dThd starvation of a TS-deficient murine cell line produces much smaller DNA fragments, 50 to 200 kb in length.99

Inhibition of protein synthesis by cycloheximide within 8 hours of FdUrd exposure dramatically reduces DNA double-strand breakage and lethality in murine FM3A cells, suggesting that FdUrd exposure triggers the synthesis of an endonuclease capable of inducing DNA strand breaks.90 In rat prostate cancer cells with intact programmed cell-death pathways, FdUrd induces oligonucleosomal fragmentation of genomic DNA.101

Factors that regulate recognition of DNA damage and apoptosis contribute to 5-FU lethality. The oncogene p53 plays a pivotal role in the regulation of cell-cycle progression and apoptosis and influences the sensitivity of murine embryonic fibroblasts to 5-FU.102 Transfection and expression of the bcl-2 oncogene in a human-lymphoma cell line renders it resistant to FdUrd. TS inhibition, dTTP depletion, and induction of single-strand breaks in nascent DNA are similar in vector control cells and bcl-2-expressing cells.103 In vector control cells, induction of double-stranded DNA fragmentation in parental DNA coincides with onset of apoptosis. The contribution of DNA damage to cell lethality varies among different malignant lines, and DNA fragmentation does not appear to contribute to 5-FU-mediated cytotoxicity in some cancer cell lines.104, 105

In summary, TS inhibition, as seen in “pure” form with FdUrd treatment in the absence of dThd salvage, and 5-FU incorporation into RNA are capable of producing lethal effects on cells. DNA damage also contributes to cytotoxicity and can occur in the absence of detectable FdUTP incorporation into DNA. The combined effects of deoxyribonucleotide imbalance (high dATP, low dTTP, high dUTP) and misincorporation of FdUTP and dUTP into DNA result in a number of deleterious consequences affecting DNA synthesis and the integrity of nascent DNA. The pattern and extent of DNA damage induced by fluoropyrimidines in human colorectal cancer cells varies and may be affected by the activity of enzymes involved in DNA repair and by downstream pathways that are required to implement cellular destruction. It is now recognized that the genotoxic stress resulting from TS inhibition activates programmed cell-death pathways, resulting in induction of parental DNA fragmentation. Depending on the cell line in question, two different patterns of parental DNA damage may be noted: internucleosomal DNA laddering, the hallmark of classical apoptosis, and high-MW DNA fragmentation with segments ranging from approximately 50 kb to 1 to 3 megabases. Differences in the type and activity of endonucleases and DNA-degradative enzymes triggered in a given cell line most likely explain these disparate patterns of parental DNA fragmentation. In “apoptosis-competent” cancer cell lines, such as HL60 promyelocytic leukemia cells, genotoxic stress results in rapid (within hours) induction of programmed cell death, with classic DNA laddering. In contrast, many cancer cell lines derived from epithelial tumors, including colon cancer, appear to undergo delayed programmed cell death. This phenomenon may reflect a “postmitotic” cell death, in which one or more rounds of mitosis are needed before cell death occurs.106 In such cell lines, the duration of the genotoxic insult may determine whether induction of cytostasis or programmed cell death occurs. One possible explanation for delayed apoptosis is that originally sublethal damage to genes, which are essential for cell survival, may ultimately lead to cell death with subsequent rounds of DNA replication.

Factors operating downstream from TS clearly influence the cellular response to genotoxic stress, such as overexpression of the cellular oncoproteins bcl-2 and mutant p53. Disruption of the signal pathways that sense genotoxic stress or lead to induction of programmed cell death, or both, may render a cancer cell inherently resistant to 5-FU. In some cancer cell lines, thymine-less death may be mediated by Fas and Fas-ligand interactions.107, 108 Fas is a cell-surface receptor that belongs to the tumor necrosis factor-receptor superfamily. Binding of Fas by Fas-ligand activates caspase 8, thus initiating a proapoptotic cascade. Cancer cell lines that are insensitive to Fas-mediated apoptosis are insensitive to 5-FU, suggesting that modulation of their expression may influence sensitivity to 5-FU.107, 108, 109, 110 Although induction of programmed cell death is generally thought to be a consequence of DNA-directed events, 5-FU-mediated induction of apoptosis in intestinal crypt cells with an intact p53 pathway appears to be a consequence of RNA-directed effects.111 Although induction of apoptosis is the final common pathway for cell death, DNA- and RNA-directed effects of 5-FU may provide the triggering stimulus.

As previously mentioned, base excision repair plays an essential role in removing incorporated 5-FU and uracil residues from DNA, resulting in single-strand DNA breaks. Because BER involves mutliple proteins, deficiencies in one of the components such as uracil DNA glycosylase, XRCC1 or DNA polymerase-β, may negatively modulate the toxic effects of TS inhibitors.112

Microsatellite instability (MSI) is a manifestation of genomic instability in human cancers that have a decreased overall ability to faithfully replicate DNA, and is a surrogate phenotypic marker of underlying functional inactivation of the human DNA mismatch repair genes (MMR).113 Functional loss of a MMR gene results from inactivation of both alleles via some combination of coding region mutations, loss of heterozygosity, and/or promoter methylation, which leads to gene silencing. In vitro studies suggest that MMR-proficient cells are more sensitive to 5-FU or FdUrd than MMR-deficient cells.114, 115 The MSI phenotype has been associated with a better prognosis in stage-for-stage matched tumors in primary colorectal cancer,116 but data are conflicting as to whether MSI status influences benefit from 5-FU-based adjuvant therapy.

Relative Importance of RNA- versus DNA-Directed Effects

The relative contributions of DNA- and RNA-directed mechanisms to the cytotoxicity of 5-FU are influenced by the specific patterns of intracellular drug metabolism, which vary among different healthy and tumor tissues. 5-FU concentration and duration of exposure play pivotal roles in determining the basis of cytotoxicity. The improved response rates observed with LV modulation of bolus 5-FU therapy, the correlation between high TS expression in tumor tissue and insensitivity to 5-FU-based therapy, and the clinical activity of the antifolate-based TS inhibitors provide strong evidence that TS is an important therapeutic target. In some models, RNA-directed effects have been predominant, with prolonged duration of exposure, and are not necessarily cell-cycle dependent, whereas DNA-directed effects have been important during short-term exposure of cells in S phase. In different models, contrary results have been observed. The mechanism of insensitivity differs in human colon cancer cells selected for resistance to either short-term, high-concentration 5-FU exposure (1,000 µmol/L for 4 hours, simulating bolus administration) or more prolonged, lower-concentration exposure (15 µmol/L for 7 days).117 A subline resistant to short-term 5-FU exposure has decreased 5-FU-RNA incorporation, whereas the subline insensitive to protracted 5-FU exposure displays more rapid recovery from TS inhibition after drug exposure. The subline with RNA-directed resistance retains sensitivity to protracted exposure.118

In two human colon carcinoma cell lines, the determinants of cytotoxicity with prolonged (120-hour) exposure to 5-FU at pharmacologically relevant concentrations (0.1 to 1.0 µmol/L) suggested that DNA-directed effects (inhibition of TS and induction of single-strand breaks in nascent DNA) and the gradual and stable accumulation of 5-FU into RNA both contribute to 5-FU toxicity.119 Thus, the primary mechanism of 5-FU cytotoxicity varies among cancer cell lines and can change within a given cell line by alterations in schedule or the circumstances of drug exposure (the presence or absence of potential modulators of toxicity). More than one mechanism of action may be operative, and each may contribute to cytotoxicity.

DETERMINANTS OF SENSITIVITY TO FLUOROPYRIMIDINES

Because of the complexity of fluoropyrimidine metabolism and the multiple sites of biochemical action, multiple factors may be associated with responsiveness to this class of antimetabolites (Table 7-4). Deletion of or diminished activity of the various activating enzymes may result in resistance to 5-FU.120, 121, 122,123, 124, 125, 126, 127, 128, 129 Conversely, elevated levels of certain activating enzymes have been associated with increased fluoropyrimidine sensitivity. Clones derived from murine leukemia selected for stable resistance to either 5-FU, FUrd, or FdUrd is each deficient in one enzyme involved in pyrimidine metabolism: decreased OPRT was associated with 5-FU resistance, whereas FdUrd and FUrd resistance was associated with deletion of dThd and Urd kinase, respectively.122, 123 Clones retained sensitivity to alternate fluoropyrimidines; thus, resistance to 5-FU may not preclude sensitivity to FdUrd, or vice versa.

TABLE 7.4 DETERMINANTS OF SENSITIVITY TO 5-FLUOROURACIL (5-FU)

Extent of 5-FU anabolism
   Cellular uptake FUrd and FdUrd require facilitated nucleoside transport)
   Activity of anabolic enzymes
   Availability of (deoxy)ribose-1-phosphate donors and phosphoribosyl phosphate
Activity of catabolic pathways
   Alkaline and acid phosphatases
   Dihydropyrimidine dehydrogenase
Thymidylate synthase (TS)
   Baseline activity of enzyme
   Affinity of TS for fluorodeoxyuridine monophosphate
   Stability of the ternary complex
   Intracellular reduced-folate content
   Transport across cell membranes
   Polyglutamation
      Folylpolyglutamate synthetase activity
      Folylpolyglutamate hydrolase activity
   Concentration of deoxyuridine monophosphate
   Up-regulation of TS protein expression with TS inhibition
Extent of fluorouridine triphosphate incorporation into RNA concentration of competing normal substrates (uridine triphosphate, cytidine 5′ triphosphate)
Salvage pathways
   Thymidine rescue
   Uridine rescue
Extent of deoxyuridine triphosphate (dUTP) and FdUTP incorporation into DNA
   Ability to accumulate dUTP (dUTP hydrolase activity)
   Uracil-DNA-glycosylase activity
Activity of other enzymes involved in base-excision repair
Extent and type of DNA damage
   Single-strand breaks
   Double-strand breaks
   Newly synthesized DNA vs. parental DNA
   Activity of DNA repair enzymes
Cellular response to genotoxic stress
   Cytostasis vs. cell death
   Intact DNA damage recognition pathways
   Intact programmed cell death signaling pathways
   Duration of genotoxic stress

FdUrd, 5-fluoro-2′-deoxyuridine; FUrd, 5-fluoro-uridine.

In addition to the importance of these activating enzymes, the availability of ribose-1-phosphate, dR-1-P, and PRPP may influence activation and response.8, 9,130, 131, 132, 133, 133


Inosine and deoxyinosine augment 5-FU activation to the ribonucleotide and deoxyribonucleotide levels by serving as a source of ribose-1-phosphate and dR-1-P.

The formation of 5-fluoropyrimidine nucleotides within target cells and the size of the competitive physiologic pools of UTP and dTTP also influence 5-FU cytotoxicity.134, 135, 136, 137, 138 The extent of 5-FU incorporation into RNA depends on FUTP formation and the size of the competing pool of UTP. Strategies that increase FUTP formation generally increase incorporation of FUTP into RNA and enhance 5-FU toxicity. Modulators including 6-methylmercaptopurine riboside (MMPR), N-phosphonoacetyl-l-aspartic acid (PALA), pyrazofurin, MTX, and dThd may increase FUTP formation by virtue of inhibiting de novo purine or pyrimidine synthesis, thereby increasing PPRP levels. Through feedback inhibition, expansion of dTTP pools decreases FdUMP formation by two means: blocking phosphorylation of FdUrd by dThd kinase and inhibiting the reduction of FUDP to FdUDP. In contrast, expansion of UTP or cytidine triphosphate (CTP) pools inhibits formation of FUMP by Urd kinase. Changes in nucleotide pool size have been implicated in 5-FU resistance in Chinese hamster fibroblast and in murine S49 lymphoma sublines that have altered CTP synthase activity, increased CTP pools, and decreased UTP pools.139, 140

Because RNA- and DNA-directed effects of 5-FU may differ in importance among different malignant cell lines, any single manipulation of 5-FU metabolism may produce conflicting results if different tumor models are compared. The development and application of sensitive assays that permit reliable measurement of FUTP, 5-FU-RNA levels, and TS inhibition in patient samples will help elucidate clinical determinants of sensitivity to fluorinated pyrimidines given by various schedules. RNA and DNA incorporation in tumor biopsy specimens taken 2, 24 or 48 hours from patients receiving bolus 5-FU (500 mg/m2) was measured using gas chromatography/mass spectrometry after complete degradation of isolated RNA and DNA to bases. Maximal incorporation occurred 24 hours after 5-FU administration: 1.0 pmol/mg RNA (n= 59) and 127 fmol/mg DNA (n= 46). Incorporation into RNA, but not DNA, significantly correlated with intratumoral 5-FU levels. The extent of TS inhibition, but not RNA or DNA incorporation, correlated with response to 5-FU therapy.141 Results of such studies in clinical samples from patients receiving various infusional schedules of 5-FU are not yet available.

Determinants of Thymidylate Synthase Inhibition

The ability of FdUMP to inhibit TS is influenced by several variables, including the concentration of enzyme, the amount of FdUMP formed and its rate of breakdown, the levels of the competing healthy substrate (dUMP) and 5,10-CH FH4 cofactor, and the latter's extent of polyglutamation. The degree and persistence of TS inhibition is a crucial determinant of cytotoxicity. Blockade of TS can lead to a gradual expansion of the intracellular dUMP pool; resumption of DNA synthesis is a function of two factors: the rate of decrease of intracellular FdUMP and the rate of increase in dUMP, which competes with FdUMP for newly synthesized TS and for enzyme that has dissociated from the ternary complex.

FdUMP accumulates rapidly in both responsive L1210 leukemia and resistant Walker 256 carcinoma, but more rapid recovery of DNA synthesis in the insensitive line correlates with accelerated decline in intracellular free FdUMP concentrations.134 Other studies have confirmed that a more rapid decline in FdUMP concentration may be characteristic of resistant neoplasms, perhaps because of the increased phosphatase activity.117, 120, 135 The basis for resistance in some cells may be explained by the rate of nucleotide inactivation rather than slower formation of the active product.

Determination of TS content in tumor tissue may help to clarify the relationship between pretreatment TS levels and prognosis, response, or both, to 5-FU therapy. Biochemical assays permit measurement of dUMP, TS, the ternary complex, and free FdUMP.136, 137, 138, 142, 143 The total content of TS is estimated by the [3H]FdUMP-binding assay. TS catalytic activity is determined by a tritium release assay (using either [5-3H]dUrd in intact cells or [5-3H]dUMP in cytosolic preparations); during dTMP formation, the addition of a methyl group displaces the [3H] from the carbon-5 of dUMP. Although these assays are extremely useful for preclinical studies, their application to clinical tumor samples is limited by the need for relatively large quantities of tissue (at least 50 mg) as well as fresh or frozen tumor tissue. Despite the limitations, these biochemical assays have yielded important information. Biopsies of liver metastases obtained 20 to 240 minutes after 500 mg/m2 5-FU among 21 patients undergoing elective surgery, maximal TS inhibition occurred within 90 minutes and averaged 70 to 80% in tumor tissue.144 Large variations in TS binding and catalytic activity were noted in primary colon tumors, but the overall enzyme levels were significantly higher than in adjacent healthy colonic tissue.145

Measurement of TS gene expression provides an alternative to directly assaying intracellular TS enzyme. Polymerase chain reaction (PCR)-based methods can quantitate the expression of TS in clinical tumor samples, and overexpression to TS in tumor biopsies correlates with insensitivity to 5-FU-based regimens.146,147, 148

Monoclonal antibodies have been developed that are capable of detecting human TS in immunoprecipitation and enzyme-linked immunosorbent assays (ELISA) and by immunoblot analysis, which have high specificity and tight binding affinities.149 Immunologic quantitation of TS in 10 5-FU-sensitive and resistant cell lines showed a good correlation with biochemical assays; the limit of sensitivity was 0.3 fmol protein in lysates.150 TS protein content in 1-mg tumor biopsy specimens can be measured with an ultrasensitive ELISA and chemiluminescent technique with a lower limit of detection of 30 amol.151 A number of studies have reported a relationship between TS expression in clinical specimens and prognosis; a systematic review of such studies in colorectal cancer has been published.152

Quantitative and qualitative changes in TS have been identified in cells with innate or acquired resistance to fluoropyrimidines. Amplification of the TS gene, with corresponding elevation of enzyme content, has been found in lines resistant to 5-FU or FdUrd.153, 154, 155 Resistant cell lines may have an altered TS protein with either decreased binding affinity for FdUMP or decreased affinity for 5,10-CH FH4.156, 157, 158, 159 Error-prone PCR has been used to mutagenize the full-length human TS cDNA and then to selected mutants resistant to FdUrd. Mutations distributed throughout the linear sequence and three-dimensional structure of human TS, including those distant from the active site, conferred resistance.160 Decreased stability of the ternary complex has been described in HCT 8 cancer cells with acquired resistance to 5-FU and LV.161 The rate of LV uptake, expansion of the reduced-folate pool, and polyglutamate chain length distribution of 5,10-CH FH4 were similar in both lines, suggesting that mutations in TS may account for the reduced formation and stability of the ternary complex.

Adequate reduced-folate pools are required to form and maintain a stable ternary complex. Administration of exogenous reduced folates enhances the cytotoxicity of 5-FU and FdUrd in preclinical models, and clinical administration of LV is used to elevate the reduced-folate content in the cancer cell.162 Tumor cells transport LV intracellularly and convert the folates to more potent and stable polyglutamates.163, 164, 165, 166 Deficiency of the low-affinity, high-capacity folate transport system (impaired membrane transport) and reduced folylpolyglutamate synthetase activity (impaired polyglutamation) impair the ability of LV to expand the reduced-folate pools.

dTTP depletion after fluoropyrimidine exposure influences sensitivity; salvage of preformed dThd by dThd kinase can bypass FdUMP-mediated TS inhibition and represents a potential mechanism of resistance.167, 168 Coadministration of 5-FU with an inhibitor of nucleoside transport would theoretically prevent cellular entry of preformed dThd. In a human colon parental line (GC C) and a subline selected for dThd kinase deficiency, the cytotoxicity and cellular pharmacology of 5-FU were similar, although only the parental line could be rescued by exogenous dThd.169

In summary, to inhibit TS, 5-FU must reach the tumor and then be metabolized to FdUMP. Cell lines lacking the capacity for nucleoside transport are unresponsive to FdUrd but retain sensitivity to 5-FU.170, 171 Additional factors influence the ability of FdUMP to inhibit TS. The tumor cell must enter the vulnerable synthetic phase of the cell cycle during drug exposure. The intracellular reduced-folate content must be adequate to promote stable inhibition of TS. The ratio of endogenous dUMP to FdUMP pools can affect the duration of TS inhibition. In certain cell lines, however, dUMP accumulation is associated with increased formation of dUTP; incorporation of dUTP into DNA may subsequently contribute to cytotoxicity by enhancing DNA damage.

Regulation of Thymidylate Synthase

TS is required for DNA replication; its activity is higher in rapidly proliferating cells than in noncycling cells. When nonproliferating cells are synchronized and stimulated to enter the synthetic phase of the cell cycle, TS content may increase up to 20-fold.172 In proliferating cancer cells, TS activity varies by fourfold to eightfold from resting to synthetic phase.173 Increased expression of the TS gene at the G1-S boundary is controlled by posttranscriptional regulation; elements in the promoter region of the human TS gene may also regulate gene expression.174, 175

5-FU exposure may be accompanied by an acute increase in TS content, which may in turn permit recovery of enzymatic activity, and the magnitude of the increase is influenced by drug concentration and time of exposure.176, 177, 178, 179, 180, 181, 182 In NCI-H630 colon cancer cells, TS content increases up to 5.5-fold during 5-FU exposure and is regulated at the translational level.181 TS protein binds to specific regions in its corresponding TS-mRNA, which contributes to the regulation of TS-mRNA translation.182, 183 Antisense oligodeoxynucleotides targeted at the AUG translational start site of TS-mRNA inhibit translation in rabbit reticulocyte lysate; transfection of KB31 nasopharyngeal cancer cells with a plasmid construct containing the TS antisense fragment decreases the expression of TS protein and enhances the sensitivity to FdUrd by eightfold.184 Small interfering double-stranded RNA (siRNA) targeted against TS are effective inhibitors of TS protein expression, and may have therapeutic potential by themselves or in combination with TS inhibitor compounds.185

Reduced-folate content also influences TS expression. A functionally TS-negative mutant (TS-C1) that has normal levels of TS-mRNA transcripts and immunologically reactive TS protein has normal clonogenic growth in the presence of high folate levels, suggesting folate responsiveness of the TS-C1 mutant.186, 187 The mutant has greatly reduced affinity for the reduced-folate cofactor, and endogenous total reduced-folate pools were only 6% of the parental level. Exposure of TS-C1 cells to 20 µmol/L LV stimulated de novo dTMP synthesis by 6 hours, whereas over 80% of the TS activity was lost by 24 hours after LV removal.187

Importance of Schedule of Administration in Preclinical Models

Drug concentration and duration of exposure in vitro are important determinants of response to 5-FU.36, 51, 188, 189, 190


High drug concentrations (above 100 µmol/L) are generally required for cytotoxicity if the duration of exposure is brief (<6 hours), whereas prolonged exposure (>72 hours) to concentrations between 1 and 10 µmol/L results produces cytotoxicity among a various tumors.

CLINICAL PHARMACOLOGY OF 5-FLUOROURACIL

The pharmacokinetics of 5-FU are important because of the choices of routes and schedules of administration available for this drug. Regional approaches permit selective exposure of specific tumor-bearing sites to high local concentrations of drug. Pharmacokinetic studies have played an important role in assessing these therapeutic alternatives.

Clinical Pharmacology Assay Methods

5-FU has been assayed in biologic fluids using high- performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). Various methods are used to extract 5-FU from biologic fluids. In general, an initial deproteination step is performed by chemical or filtration techniques. Subsequent steps separate 5-FU from other constituents in biologic fluids that may interfere with 5-FU detection. A number of different HPLC assays have been described for the analysis of 5-FU, including reversed-phase, reversed-phase ion-pairing, and normal-phase chromatography. HPLC methods using ultraviolet detection of 5-FU are typically associated with limits of detection in the range of 0.2 to 1.0 µmol/L. Column or valve-switching techniques and the use of microbore-HPLC columns can further improve the limits of detection.

The nucleoside metabolites of 5-FU can be separated from parent drug on reversed-phase and ion-exchange columns, whereas separation of the nucleotide metabolites is obtained with either anion-exchange or reversed-phase ion-pairing methods. Preclinical studies describing intracellular metabolism generally typically use radiolabeled 5-FU; HPLC with inline liquid scintillation detection is used to quantify the metabolites.

Derivitization of 5-FU is required for GC-MS. Mass spectrometry generally provides much greater sensitivity than that achievable with HPLC, with limits of detection as low as 0.5 ng/mL (4 nmol/L) for a 1-mL plasma sample.191, 192 Recent advances in fluorine-19 magnetic resonance imaging (MRI) have permitted monitoring of the pharmacokinetics and cellular pharmacology of 5-FU, thus allowing noninvasive determination of 5-FU content in tissues.193

5-FU is unstable in whole blood and plasma at room temperature, and catabolism is much more rapid in whole blood than in plasma.194, 195 Blood samples should be placed on ice immediately; plasma should be quickly isolated. 5-FU is stable in plasma at 4°C for up to 24 hours and is stable for prolonged periods when stored at -20°C.

ABSORPTION AND DISTRIBUTION

Bioavailability of 5-FU by the oral route is erratic; <75% of a dose reaches the systemic circulation.195, 196, 197 When administered by intravenous bolus or infusion, 5-FU readily penetrates the extracellular space, cerebrospinal fluid (CSF) and extracellular “third-space” accumulations. The volume of distribution (Vd) ranges from 13 to 18 L (8 to 11 L per m2) after intravenous bolus doses of 370 to 720 mg per m2, which slightly exceeds extracellular fluid space.191, 198

Plasma Pharmacokinetics

The pharmacokinetic profile of 5-FU varies according to dose and schedule of administration. After intravenous bolus injection of 370 to 720 mg per m2, peak plasma concentrations (Cp) of 5-FU are 300 to 1,000 µmol per L (Table 7-5).191, 198, 199, 200, 201 Rapid metabolic elimination accounts for a primary t1/2 of 8 to 14 minutes; 5-FU Cp fall below 1 µmol per L within 2 hours.

McDermott et al. reported triexponential elimination of intravenous bolus 5-FU with t1/2 values of 2, 12, and 124 minutes.202 A prolonged third elimination phase of 5-FU was noted by GC-MS after bolus administration with a t1/2 of 5 hours: 5-FU Cp ranged from 36 to 136 nmol per L 4 to 8 hours after intravenous bolus doses of 500 to 720 mg/m2 and may reflect tissue release.191

The clearance of 5-FU is much faster with continuous infusion (CI) than with bolus administration and increases as the dose rate decreases (Table 7-6).192, 197,203, 204, 205, 206, 207, 208 As the duration of 5-FU infusion increases, the tolerated daily dose decreases. A recommended starting dose of single-agent 5-FU given by protracted CI is 300 mg/m2; the achieved steady-state plasma levels (Css) are in the submicromolar range. With CI over 96 to 120 hours, a daily dose of 1,000 mg/m2 produces a Css in the 1 to 3 µmol/L range, and an intermittent schedule is necessary. CI of 2,000 to 2,600 mg/m2 5-FU daily given either for 72 hours every 3 weeks or for 24 hours weekly yields a Css of 5 to 10 µmol/L.

5-FU clearance varies considerably between individuals. The elimination kinetics of 5-FU are nonlinear.191, 195, 196, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214 The following are noted with increasing doses: a decrease in hepatic extraction ratio; an increase in bioavailability; an increase in plasma t1/2; a decrease in total-body clearance; and an increase in 5-FU area under the curve (AUC). Although the change in 5-FU clearance or AUC with increasing 5-FU dosage on a given schedule may be linear over a certain dose range, with higher dosages these parameters may change disproportionately. This nonlinear behavior represents saturation of metabolic processes at higher drug concentrations, leading to difficulty in predicting plasma levels or toxicity at higher dosages.

TABLE 7.5 PHARMACOKINETICS OF 5-FLUOROURACIL GIVEN BY INTRAVENOUS BOLUS

Investigators

Dose (mg/m2)

No.

Half-Life (min)

Clearance (mL/min/m2)

Plasma Concentration (µmol/L)

AUC per Dose (µmol•min/L)

Grem et al.198

370

16

8.1 ± 0.4

862 ± 24

C0: 332 ± 27
15 min: 82 ± 6
60 min: 4 ± 1

3,761 ± 286

Macmillan et al.199

400

8

11.4 ± 1.5

744 ± 145

5 min: 469 ± 85
20 min: 100 ± 20
60 min: 13 ± 6

9,885 ± 1,569

Heggie et al.200

500

10

12.9 ± 7.3

594 ± 7.3

5 min: 420 ± 102
20 min: 114 ± 52
60 min: 10 ± 11

7,125 ± 2,371

van Gröeningen et al.191

500

15

9.8 ± 2.4

558

Not stated

7,338 ± 1,708

600

18

404

12,000 ± 2,446

720

7

14.4 ± 2.5

349

16,200 ± 2,446

Grem et al.201

425

11

9.8 ± 0.5 (all doses)

743 ± 81

C0: 378 ± 46

4,401 ± 363

490

13

713 ± 28

393 ± 24

5,304 ± 227

AUC, area under the concentration time curve; C0, estimated initial concentration. Note: If either AUC or clearance was not provided, it was calculated from the following equation: intravenous dose/AUC = clearance. The molecular weight of 5-FU = 130.1.

Variation in 5-FU pharmacokinetics has been reported according to time of day. A 2.2-fold difference was noted in 5-FU Cp during a 5-day infusion of 1,000 mg/m2 per day; the peak value averaged 4.5 µmol/L at 1:00 AM, whereas the minimum value averaged 2 µmol/L at 1:00 PM.207 With CI of 300 mg/m2 per day 5-FU, the peak 5-FU Cp was 0.22 µmol/L around noon; the trough 5-FU value, 0.04 µmol/L, occurred around midnight.205 The discrepancy between the times of day at which peak and trough 5-FU levels occurred in these two studies suggests that other factors, perhaps geographic, seasonal, individual sleep and wake habits, and administration of other drugs may influence 5-FU clearance.

TABLE 7.6 PHARMACOKINETICS OF 5-FLUOROURACIL GIVEN BY CONTINUOUS INTRAVENOUS INFUSION

Investigators

Duration of Infusion

Daily Dose (mg/m2)

No.

Cpss (µmol/L)

Clearance (mL/min/m2)

Grem et al.206

Protracted

64–200

24

0.30 ± 0.04 (0.14–1.04)

3,050 ± 330

Anderson et al.192

Protracted

176–300

3

0.32 (0.05–0.57)

Not provided

Harris et al.205

Protracted

300

7

0.13 ± 0.01

Not provided

Yoshida et al.204

Protracted

190–600

19

1.15 ± 0.15 (0.08–2.40)

2,033

Petit et al.207

120 hr

450–966

7

2.6 ± 0.2

Not provided

Fleming et al.208

120 hr

1,000

57

2.1

2,523 ± 684

Fraile et al.197

96 hr

1,000–1,100

6

24–48 hr, 1.3 ± 0.1
72–96 hr, 1.8 ± 0.3

Not provided

Benz et al.203

24 hr

1,500

7

4 (1.94–5.63)

2,118 (1,235–3,471)

Erlichman et al.209

120 hr

1,250

15

3.4 ± 0.4

2,410

1,500

6

5.1 ± 1.0

1,790

1,750

14

6.4 ± 0.9

1,990

2,000

25

7.2 ± 0.7

1,910

2,250

17

7.5 ± 1.0

2,000

Remick et al.210

72 hr

1,655

6

5.4 ± 0.3

1,750 ± 105

2,875

8

13.9 ± 0.5

1,117 ± 37

Grem et al.211

72 hr

1,150–1,525

19

3.4 ± 0.5

3,011 ± 356

1,750

31

5.0 ± 0.5

2,671 ± 563

2,000

53

6.5 ± 0.9

2,651 ± 324

2,300

14

8.8 ± 1.3

2,116 ± 572

2,645

10

10.0 ± 2.1

2,247 ± 443

Grem et al.226

24 hr

2,300

12

6.6 ± 1.7

1,953 ± 453

Css, plasma concentration at steady state. Note: Plasma clearance converted from milliliter per minute assuming an average body surface area of 1.7 m2 and from milliliter per kilogram assuming a conversion factor of 37 from kg to m2.

The diurnal and interindividual variations in 5-FU pharmacokinetics suggest that, to compare and assess pharmacokinetic parameters with clinical outcome or the effect of another drug, it is important to have each patient serve as his or her own control, obtain samples for pharmacokinetic studies at the same time of day and for consecutive daily schedules, on the same day of treatment, and to collect pharmacokinetic samples for all subjects within as narrow a time window as possible.

Correlations have been described between 5-FU pharmacokinetics and toxicity with intravenous bolus and infusion schedules (Table 7-7).191, 198, 204, 206, 210,211, 215, 216, 217 Serious clinical toxicity tends to increase with higher systemic exposure, reflected by total AUC with bolus injection and Css with 5-FU infusion. These findings suggest that pharmacokinetic monitoring may be used to adjust 5-FU doses to avoid or minimize serious clinical toxicity.217, 218However, not all patients with relatively high 5-FU systemic exposure experience serious toxicity, and some patients have toxicity despite relatively low 5-FU systemic exposure, suggesting that other factors contribute. The relationship between antitumor activity and 5-FU pharmacokinetics is less clear.

FdUrd is generally given by CI. The achieved Css with protracted schedules have not been well defined because the predicted Cp are below the detection limits of HPLC assays; analysis by GC-MS has been hampered by the difficulty in preparing stable, volatile derivatives of FdUrd. With intravenous bolus FdUrd given weekly, the AUC of 5-FU is twofold to threefold greater than FdUrd, suggesting that FdUrd is acting in part as a precursor to 5-FU.219 With 1,650 mg/m2 given at the midpoint of a 2-hour infusion of 500 mg/m2 LV, the median clearance was 3,500 mL/min.

TABLE 7.7 CORRELATION OF 5-FLUOROURACIL PHARMACOKINETIC PARAMETERS WITH CLINICAL TOXICITY

Reference

Dose (mg/m2/d)

Intravenous Schedule

Parameter AUC, µmol/L/min; Css, µmol/L

Toxicity Grade

Incidence (%)

No. Patients

P Value (Test)

191

500–720

Bolus

AUC: ≤8,300
>8,300

≥1

11
71

11
28

Not stated

198

370

Bolus

AUC: <4,000
4,000–5,000
>5,000

≥ 3

0
21
43

15
14
7

.03 (Wilcoxon rank sum)

204

190–360

Protracted CI

Css: 0.8 ± 0.4a
1.5 ± 0.7a

≤2
≥ 3

100
100

9
10

<.05 (Bonferroni)

136 a

64–200

Protracted CI

Css: 0.24 ± 0.02a

# 1

100

19

.02 (Mann-Whitney)

0.53 ± 0.14

2

100

5

 

215

1,000

120 hr CI

AUC: <1,800
≥1,800

≥1

3
78

31
32

<.01 (not stated)

211

1,150–3,500

72 hr CI

≥3

 

Gastrointestinal toxicity/ANC/platelet:

   

Css: ≤8.9
≥9.0

 

1/14/0
14/41/14

91
11

.02, .01, .007 (Fisher's exact)

216

185–3,600

72 hr CI

Css: ≤2.0
2.1–4.0

≥1

6
28

32
32

% Mucositis = 100 × (1–e–0.114Css)

137

 

 

> 4.0

 

70

50

r2 = 0.88

217 b

~1,000

96 hr CI

AUC 27,622 ± ±962a

0–2 heme toxicity

 

65

.035 (t-test)

31,451 ± 1,358a

3–4

26

AUC, area under the concentration time curve; CI, continuous infusion; Css, plasma concentration at steady state; ANC, absolute neutrophil count
aMean ± SE.
bAUC units are ng•hr/mL over 96 hr.
CI, continuous infusion; HAI, hepatic arterial infusion; LV, leucovorin CI, continuous infusion; HAI, hepatic arterial infusion.

Regional Administration of 5-Fluorouracil

The administration of 5-FU and FdUrd by intrahepatic arterial infusion (HAI) is a strategy to maximize the regional exposure while limiting systemic toxicity. Approximately 19 to 51% of infused 5-FU is cleared in its first pass through the liver, whereas FdUrd first-pass clearance exceeds 94%.212 Systemic and hepatic metabolic clearances and extraction ratios decrease progressively with increasing 5-FU dose rates. Systemic exposure to 5-FU after HAI ranges from 12 to 52% of that after intravenous administration of dose rates equivalent to 0.37 to 10 g/m2; the regional advantage relative to systemic exposure varies from sixfold at the lowest 5-FU doses to twofold at the highest doses.214 Drug dose, blood flow, and the rate of administration influence the extent of hepatic removal and systemic exposure.220

Portal venous perfusion was based on the premise that although most large metastases obtain their blood supply predominantly from the arterial circulation, small metastases may be fed by the portal circulation. Mean tumor uptake of FdUrd in patients with established metastases was 15.5-fold greater after bolus administration of [3H]FdUrd into the hepatic artery compared with portal vein, whereas the uptake into healthy liver is similar.221 These findings suggest that portal perfusion may be effective in the setting of micrometastatic disease, and this approach has been explored as adjuvant therapy for stage II and III colon cancer.

Low MW compounds such as 5-FU and FdUrd injected into the peritoneal cavity are absorbed primarily through the portal circulation, passing through the liver before reaching the systemic circulation. The rates of absorption and clearance from the peritoneal cavity depend on the drug's lipid solubility and MW, as well as the surface area of the peritoneum (which may be altered by tumor, adhesions, or other pathologic changes).

Peritoneal dialysate concentrations up to 5 mmol/L 5-FU maintained by intermittent exchanges of fluid are tolerated for up to 5 days.222, 223 Mean 5-FU clearance from the peritoneal cavity was 840 mL/min, about fivefold slower than systemic clearance; the ratio of intraperitoneal to systemic 5-FU levels was 300.222 Higher intraperitoneal drug concentrations (>5 mmol/L) saturate hepatic clearance mechanisms, with increased systemic levels and significant myelosuppression. Mild-to-moderate abdominal pain and chemical peritonitis may occur, particularly with repeated dosing. 5-FU given.intraperitoneally in escalating concentrations for 4 hours along with a fixed dose of cisplatin (90 mg/m2) every 28 days was associated with dose-limiting neutropenia with 5-FU concentrations of >20 mmol/L; other toxicities included nausea, vomiting and diarrhea.224 Between dialysate concentrations of 5 and 24 mmol/L, the mean peritoneal levels of 5-FU ranged from 2.2 to 12.5 mmol/L, and peak Cp, which occurred 1 hour after intraperitoneal instillation, ranged from 6 to 60 µmol/L.

FdUrd 3 g given intraperitoneal in 2 L of 1.5% dialysate with a dwell time of up to 3 days was associated with excellent local tolerance; the major systemic toxicities, nausea and vomiting, were well controlled with antiemetics.225 Peritoneal FdUrd levels over the initial 4 hours were above 1,000 µg/mL (4 mmol/L); peritoneal 5-FU levels were also high 0.75 to 1.50 mmol/L. FdUrd Cp were below 2 µmol/L, whereas 5-FU Cp were much higher: 150 to 300 µmol/L. The AUC values extrapolated to infinity at the recommended dose indicate a pharmacologic advantage for FdUrd of about 2,700. The t1/2 and clearance of FdUrd in peritoneal fluid was 97 minutes and 31 mL/min, compared with values of 7.5 minutes and 7,000 mL/min after 2 g FdUrd intravenous over 30 minutes.225

Figure 7.5 Catabolism of 5-fluorouracil (5-FU). DHFU, dihydrofluorouracil; FBAL, fluoro-β-alanine; FUPA, α-fluoroureido-propionic acid; NADP, nicotinamide adenine dinucleotide; NADPH, nicotinamide adenine dinucleotide phosphate.

Topical 5-FU, 2 to 5% in a hydrophilic cream base or propylene glycol, is used by dermatologists for the treatment of multiple actinic keratoses of the face, intraepidermal carcinomas, superficial basal cell carcinoma, vaginal intraepithelial neoplasia, and genital condylomas. Local application of 5-FU has also been used after trabeculectomy in patients with uncontrolled glaucoma to improve intraocular pressure control.

Mechanisms of Drug Elimination

After bolus dosing of 5-FU, about 90% is eliminated by metabolism (catabolism > anabolism), and less than 10% is renally excreted.200 With continuous infusion of 5-FU 2.3 g/m2, less than 2% of 5-FU was excreted in the urine.226 The initial rate-limiting step in 5-FU catabolism is reduction of the pyrimidine ring by dihydropyrimidine dehydrogenase (DPD) (Fig. 7-5). In the presence of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), DPD converts pyrimidines, such as uracil and 5-FU, to the dihydropyrimidine form (dihydrouracil and dihydrofluorouracil [DHFU]). Kinetic studies of purified DPD from a number of mammalian tissues have shown that the affinity of uracil and 5-FU are similar (Km ranging from 1.8 to 5.5 µmol/L).227, 228, 229, 230Saturation of DPD accounts for the dose-dependent pharmacokinetics. DPD is widely distributed in tissues throughout the body, including the liver, GI mucosa, and peripheral blood mononuclear cells (PBMCs).231, 232 Because of the size of the organ, the liver has the highest total content of DPD in the body and is a major site of 5-FU catabolism.

The clearance of 5-FU during CI exceeds hepatic blood flow (1,000 mL/min) by several-fold, suggesting that a substantial portion of 5-FU metabolism occurs in extrahepatic tissue. In clinical practice, the dosage of 5-FU is not usually reduced in the presence of hepatic dysfunction. Full-dose 5-FU has been given by HAI to patients with extensive liver replacement and jaundice; improvement or resolution of jaundice may occur in some patients without undue systemic toxicity.233, 234 Patients with severe hepatic dysfunction in general have been excluded from randomized trials of HAI FdUrd and systemic 5-FU. The dosage of 5-FU need not be reduced automatically for hepatic dysfunction; however, a more conservative approach may be prudent in jaundiced patients with a poor performance status.

DHFU appears rapidly after intravenous bolus 5-FU. The pyrimidine ring is subsequently opened by dihydropyrimidinase forming “-fluoroureido-propionic acid (FUPA); FUPA is then converted by β-alanine synthase to fluoro-β-alanine (FBAL), with the release of ammonia from the nitrogen-3 position and CO from the carbon-2 of the pyrimidine ring. In 10 patients receiving 500 to 700 mg/m2 [3H]5-FU, peak DHFU Cp of 10 to 30 µmol/L were seen 30 to 90 minutes later. FUPA reached maximum Cp (Cmax) of 13 µmol/L at 90 minutes; FBAL Cmax occurred between 60 to 90 minutes (60 µmol/L). The excretion of unchanged drug, FBAL, and FUPA in the urine occurred within the 6 hours; FBAL was the major metabolite.200

Significant biliary concentrations of 5-FU (11 to 259 µmol/L) have been detected during the first hour after intravenous bolus.200 A conjugate of FBAL and cholic acid was seen within 30 minutes; peak values of 1 mmol/L were seen within 2 to 4 hours.200, 235

After intravenous bolus administration of [3H]FBAL in rats, tissue levels of FBAL were at least fivefold to 10-fold higher than the corresponding Cp for up to 8 days.236 The radioactivity was present as free FBAL in all tissues except liver, in which FBAL undergoes enterohepatic circulation as FBAL-bile acid conjugates. FBAL accumulated in tissues that correspond to sites of 5-FU toxicity in humans, suggesting it might contribute to host toxicity.

Dihydropyrimidine Dehydrogenase Activity and 5-Fluorouracil-Associated Toxicity

Human DPD protein has been purified and its crystal structure has been elucidated.229, 230, 237 DPD enzymatic activity has often been measured by incubating cellular lysates with excess NADPH and radiolabeled 5-FU, with separation of 5-FU and DHFU by HPLC and scintillation detection. This is a labor-intensive assay that has generally limited its availability to research laboratories. Although there appears to be a relationship between DPD activity and 5-FU clearance, the correlation is not tight in general study populations of cancer patients that have not been preselected for insensitivity to 5-FU-based therapy.205, 208, 238In contrast, profound DPD deficiency is more likely to be identified in patients who have experienced excessive toxicity with a 5-FU-based therapy.239, 240Population studies that measured DPD activity in human peripheral blood mononuclear cells suggest a Gaussian distribution.238, 239, 241, 242, 243, 244, 245Although total DPD deficiency is relatively rare, a cut-point of 100 pmol/min per milligram may designate patients with partial DPD deficiency at increased risk of toxicity with 5-FU-based therapy.239, 241 The wide interpatient variability in DPD activity in these studies is consistent with the broad interpatient variations in 5-FU clearance.

The human cDNA for DPD has been cloned, and the gene is localized to the centromeric region of human chromosome 1 between 1p22 and q21.246, 247 The structural organization of the human DPD gene indicates that it is approximately 150 kb in length and consists of 23 exons ranging in size from 69 to 1,404 base pairs.248 Given the large size of the DPD protein, several different molecular defects have been described in different populations of DPD-deficient kindreds, including point mutations and deletions caused by exon skipping.249, 250, 251, 252 Familial studies suggest that total DPD deficiency is associated with an autosomal-recessive pattern of inheritance. However, childhood familial thymine-uraciluria in homozygous-deficient patients has a variable clinical phenotype, and not all subjects exhibit the abnormal phenotype.251, 253

Pharmacokinetic interactions between 5-FU and several compounds are related to interference with 5-FU catabolism by DPD. Clinical studies using pharmacologic doses of dThd with 5-FU demonstrated marked slowing of 5-FU clearance.254, 155, 256 The clearance of 5-FU was inversely related to the plasma level of thymine, which competitively inhibits the catabolism of 5-FU by DPD.255 Pyrimidine nucleosides and bases competitively interfere with the catabolism of various substrates by DPD.257 Chronic cimetidine therapy (1,000 mg daily in divided doses for 4 weeks) has been reported to decrease the clearance of 5-FU (555 mg/m2 of intravenous bolus) by 28%.258 Studies in rats and monkeys indicate that chronic cimetidine treatment decreases the clearance of 5-FU, apparently as a result of inhibition of DPD activity.259 Cimetidine is an H2-receptor antagonist; of note, ranitidine is chemically distinct and does not affect 5-FU clearance.

In Japan, shortly after the commercial release of 1-β-d-arabinofuranosyl-(E)-5-(2-bromovinyl) uracil (sorivudine), an oral antiviral agent with activity against herpes zoster, 15 patients died, and other patients experienced severe clinical toxicity while taking concomitant oral 5-FU prodrugs.260, 261, 262 The basis for this interaction was shown to be production of (E)-5-(2-bromovinyl)uracil (BVU) by gut flora.260 In the presence of NADPH, BVU forms a covalent complex with DPD, thereby inhibiting its activity. Rats treated with the combination of oral ftorafur and sorivudine had markedly elevated levels of 5-FU in plasma and in tissues; all animals died within 10 days with marked myelosuppression, atrophy of intestinal membrane mucosa, bloody diarrhea, and severe anorexia, which mirrored the clinical picture. Rats given sorivudine or ftorafur alone had minimal toxicity. Prolonged inhibition of DPD for up to 19 days has been documented in patients with herpes zoster taking sorivudine (40 mg once daily for 10 days).261 Sorivudine is an investigational antiviral agent in North America and Europe. Patients receiving sorivudine should not receive other fluoropyrimidines for at least 4 weeks after completing sorivudine therapy and should be monitored carefully thereafter.262

Impact of Schedule on Clinical Toxicities

The main toxic effects of 5-FU and FdUrd occur in rapidly dividing tissues (primarily gastrointestinal mucosa and bone marrow). The spectrum of toxicity associated with 5-FU and FdUrd (Tables 7-8 and 7-9) varies according to dose, schedule, and route. In general, bolus administration produces more myelosuppression than infusional schedules. The toxicity of infusional 5-FU depends on dose and duration.

A 5-day loading course (10 to 15 mg/kg per day) of intravenous bolus 5-FU followed by half-dosages every other day for 11 dosages or until toxicity supervened caused a 3% mortality rate.263, 264 Modification to a 5-day loading course followed on recovery by single weekly dosages was associated with leukopenia, mucositis, nausea and vomiting, diarrhea, and dermatitis. Bolus FdUrd given daily for 5 days (30 mg/kg per day) followed by half-dosages every other day for 11 dosages or until toxicity supervened led to a similar toxicity profile to bolus 5-FU except for a higher incidence of nausea, vomiting and dermatitis.263 Mucositis and diarrhea are dose-limiting with bolus 5-FU given intravenous daily for 5 days every 4 weeks, although neutropenia may also be problematic.265 A single intravenous bolus dose given weekly is frequently associated with myelosuppression, diarrhea, and mucositis.226

TABLE 7.8 RELATIONSHIP OF ROUTE AND SCHEDULE TO TOXICITY OF 5-FLUOROURACIL

Reference

Route/Schedule

Daily Dose: mg/m2(Exceptions Noted)

Toxicities

265, 273, 275

IV bolus daily for 5 days q 4 weeks

500
425 (+LV 20)
370–400 (+LV 200)

Myelosuppression, mucositis, diarrhea Ocular, dermatitis

266

IV bolus weekly (for 6 of 8 weeks)

750
500–600 (+LV 500/2-hr)

Myelosuppression, diarrhea mucositis, ocular

270,271,272

IV CI 24 hr q week

2,600
2,300–2,600 (+LV 50–500/24 hr)

Neurologic, diarrhea
Mucositis, skin (hand-foot), myelosup-pression

273

IV CI 48 hr q week

1,750/24 hr (3,500 total)

Diarrhea, mucositis, skin (hand-foot), myelosuppression, neurologic

210, 211

IV CI 72 hr q 3 weeks

2,300/24 hr (6,900 total)
2,000 (+LV 500/24 hr)

Mucositis
Diarrhea, myelosuppression

197, 208

IV CI over 96–120 hr q 3 weeks

1,000/24 hr (4,000–5,000 total)

Mucositis, diarrhea, myelosuppression, dermatitis

267, 268

IV CI over 144 hr q 3 weeks

750/24 hr

Mucositis, skin (hand-foot), diarrhea

270, 274

IV CI over 24 hr daily for 4 weeks with 1 week rest

300
200 (+LV 20 q week)

Skin (hand-foot), mucositis, Diarrhea, myelosuppression

275

IV bolus + CI over 22-hr day 1,2 q 2 weeks

400 + 600/22 hr (+LV 200/2 hr)

Myelosuppression, diarrhea, mucositis, conjunctivitis

276

IV CI over 48 hr q 2 weeks

1,500–2,000 (+LV 500/2 hr day 1, 2)

Diarrhea, mucositis, myelosuppression, skin (hand-foot), neurologic

277

HAI CI over 24 hr daily for 14–21 days

750–1,100

Mucositis, diarrhea, upper gastrointestinal ulceration, myelosuppression, chemical hepatitis

278

HAI IV over 15 min CI over 22-hr day 1,2 q 2 weeks

400 1,600 (+LV 200/2 hr day 1, 2)

Diarrhea, cardiac, neurotoxicity

223, 224

IP installation for 32–120 hr q 28 days

5 mmol/L

Mucositis, diarrhea, peritonitis, myelosuppression

224

IP installation over 4 hr q 28 days

3,900 mg (15 mmol/L) (+cisplatin 90 mg/m2)

Myelosuppression, nausea and vomiting, diarrhea, abdominal pain

Topical daily

5% cream

Local inflammation

CI of 5-FU has been given over durations ranging from 24 hours to several weeks. With infusion durations of 72 to 120 hours, 5-FU is generally given at 3- to 4-week intervals. The tolerated daily dosage decreases as the duration of infusion increases. Mucositis is usually dose-limiting with CI of 1000 or 750 mg/m2per day for 4 or 5 days, respectively, although diarrhea and dermatitis occur; myelosuppression is generally mild to moderate.197, 207, 267 With a 72-hour CI, 2,000 to 2,300 mg/m2 per day is tolerated.210, 211 Mucositis (18% grade 3 to 4) is dose-limiting with CI of 750 mg/m2 5-FU daily for 7 days every 3 weeks, 14% of patients experience grade 2 or worse palmar-plantar erythrodysesthesia (hand-foot syndrome).268 Intermittent doses up to 14 g (8 g/m2) over a 24-hour period have been tolerated, but this latter schedule is not currently in clinical use.269 High-dose 5-FU infusion over 24-hours repeated weekly involves 2,600 mg/m2; neurotoxicity and GI toxicity are dose-limiting.270, 271, 272 The 5-FU dosage is 1,750 mg/m2 per day when given weekly over 48 hours; diarrhea and mucositis are generally dose-limiting.273 With protracted CI of 5-FU, the recommended dosage is 300 mg/m2 per day.270, 274 When initially developed, the intention was to continue the infusion indefinitely until toxicity supervened.274 However, a daily-for-28-days schedule followed by a 1-week break is now more frequently used.270 Mucositis and hand-foot syndrome are dose-limiting, whereas diarrhea is less common.

TABLE 7.9 TOXICITY ASSOCIATED WITH VARIOUS CLINICAL SCHEDULES OF 5-FLUORO-2′-DEOXYURIDINE

Reference

Route/Schedule

LV

Maximum Daily Dose

Toxicities

mg/kg

mg/m2

263

IV bolus × 5 day × q 4 week

No

30

1,110

Diarrhea, mucositis

219

IV bolus q week × 6 q 8 week

Yes (high)

45

1,650

Diarrhea

269

IV CI × 3 days

No

30

1,100

Diarrhea, mucositis, and myelosuppression

279

IV CI × 5–7 days q 3–4 weeks

No

0.75–1.00

28–37

Mucositis, diarrhea

285

IV CI × 5 days q 3 weeks

Yes (high)

0.3

11.1

Myelosuppression

280,281

IV CI × 14 days q 4 weeks

No
Yes (low)

0.15
0.075

5.6
2.8

Diarrhea

280,281

HAI × 14 days q 28 days

No

0.125–0.150

7.4–11.0

Chemical hepatitis, cholestatic jaundice, and biliary sclerosis, upper GI ulceration

225

i.p. 4 hr day × 3 q 3 weeks

No

 

3,000

Nausea and vomiting

CI, continuous infusion; 5-FU, 5fluorouracil; HAI, hepatic arterial infusion.

An every-2-week schedule of LV-modulated 5-FU given by combined bolus and CI was developed to exploit the potential for different mechanisms of action with bolus versus infusional 5-FU. A randomized study comparing the monthly schedule of low-dose LV and bolus 5-FU with a high-dose LV and 5-FU bolus plus CI every 2 weeks as first-line therapy of patients with metastatic colorectal cancer demonstrated a higher response rate (32.6% versus 14.4%, P= .0004) and median progression-free survival (27.6 versus 22 weeks, P= .0012) in favor of the every-2-week regimen.275 The bimonthly regimen has been modified several times.276

The highest tolerated dose of FdUrd administered as a 14-day CI is 0.125 to 0.15 mg/kg per day (4.6 to 5.6 mg/m2 per day). Diarrhea predominates, whereas mucositis is less common. Severe myelosuppression is uncommon with prolonged CI of either 5-FU or FdUrd. When considering comparable schedules of 5-FU and FdUrd, the tolerated doses of FdUrd given by CI for either 5 or 14 days are 90- and 50-fold lower, respectively.

Myelosuppression

Serious myelosuppression is more common with intravenous bolus schedules of 5-FU and FdUrd. The greatest impact is on leukocytes and neutrophils, although anemia may also be problematic. Serious thrombocytopenia occurred with the loading schedules, but is uncommon with current schedules. Serial bone marrow aspirates examined in patients undergoing loading courses of 5-FU revealed alterations in metamyelocytes as early as 24 hours after the first dose of 5-FU; megaloblastic erythropoiesis was the dominant process in the bone marrow between days 5 and 7, and recovery of the marrow to normoblastic hematopoiesis was apparent within 3 to 5 days after discontinuing 5-FU.286 Interference with conversion of dUMP to dTMP as a consequence of decreased activity of TS or DHFR formed the basis of the deoxyuridine suppression test, which was previously used to confirm the basis of the megaloblastic anemia. The acute megaloblastic changes seen with this bolus loading schedule are likely the result of inhibition of TS.

Gastrointestinal Toxicity

5-FU-associated GI toxicity can be severe and life- threatening. Mucositis may be preceded by a sensation of dryness that is followed by erythema, formation of a white, patchy membrane, ulceration, and necrosis. Similar lesions have been observed throughout the GI tract and in the stoma of colostomies. Enteric lesions may occur at any level, resulting in clinical symptoms of dysphagia, retrosternal burning, watery diarrhea, abdominal pain, and proctitis. The diarrhea can be bloody. Nausea, vomiting, and profuse diarrhea can lead to marked dehydration and hypotension. Disruption of the integrity of the gut lining may permit access of enteric organisms into the bloodstream, with the potential for overwhelming sepsis, particularly if the neutrophil nadir coincides with diarrhea. Radiographic changes on small bowel series have shown extensive or segmental narrowing of the ileum and thickening or effacement of the mucosal folds in the distal ileum.287

Before each dose, it is essential to question whether the patient has experienced mouth soreness, watery stools, or both. 5-FU should be withheld in cases of ongoing mucositis or diarrhea, even if mild, and subsequent dosages should be reduced when the patient has fully recovered. If diarrhea occurs, supportive care and vigorous hydration should be given as dictated by the severity of the toxic reaction. Antidiarrheal agents may provide symptomatic relief from secretory diarrhea. Loperamide is a standard therapy for uncomplicated diarrhea, but it is less effective in the setting of severe diarrhea. Aggressive management of complicated diarrhea requires intravenous fluids, octreotide, administration of antibiotics, and stool workup for blood, fecal leukocytes, and infectious causes of colitis.288

An oral hygiene program including chlorhexidine may be used to help reduce the severity of mucositis, and topical preparations such as anesthetics can provide local pain relief. A randomized, double-blind crossover study of allopurinol mouthwash in patients receiving 5-FU with LV daily for 5 days showed no amelioration of mucositis.289 Mouth cooling (oral cryotherapy) with oral ice chips for 30 minutes starting immediately before bolus 5-FU reduces the severity of mucositis.290

Skin Toxicity

Dermatologic toxicity occurs with bolus and CI schedules.291, 292, 293 Loss of hair, occasionally progressing to total alopecia, nail changes (onycholysis and pigmentation), dermatitis, and increased pigmentation and atrophy of the skin may occur. Manifestations vary from erythema alone to a maculopapular erythematous rash. 5-FU enhances the cutaneous toxicity of radiation; reactions typically occur within 7 days of radiation. Erythema followed by dry desquamation occurs, with vesicle formation in severe cases. Photosensitivity reactions may occur and can result in exaggerated sunburn reactions, residual tanning, or both, in the distribution of sunlight exposure. Hyperpigmentation over the veins into which 5-FU has been administered also occurs. Allergic contact dermatitis may occur with topical 5-FU. Actinic keratoses may develop an erythematous inflammatory reaction with systemic 5-FU. Hand-foot syndrome is particularly common with CI schedules. Oral pyridoxine 50 to 150 mg daily and liberal application of lanolin-containing creams are often suggested to ameliorate this toxicity, but definitive data are lacking.

Neurotoxicity

5-FU may produce acute neurologic symptoms. A cerebellar syndrome has been most frequently reported and may be accompanied by ataxia, global motor weakness, bulbar palsy, bilateral oculomotor nerve palsy, and upper motor neuron signs.294, 295, 296, 297, 298, 299 Serious cognitive impairment, such as somnolence, coma, organic brain syndrome, and dementia, has also been seen. These symptoms are usually reversible after drug discontinuation. Neurologic toxicity has been seen on several 5-FU schedules, but is more prominent on schedules that feature high daily doses (bolus and 24- to 48-hour infusions) or with intensive daily schedules. Neurotoxicity has been prominent in some studies of 5-FU given with biomodulators including dThd, PALA, and allopurinol.270,272, 300, 301, 302

Severe neurotoxic reactions, including coma, have been reported in patients with previously unrecognized complete deficiency of DPD after receiving conventional doses of 5-FU, and the time to recovery may be longer than in non-DPD-deficient patients.303, 304, 305 Pharmacokinetic analysis of one patient confirmed a markedly prolonged t1/2 of 5-FU; no catabolites were identified in serum, urine, or CSF; the neurotoxic reactions correlated with prolonged exposure to elevated 5-FU Cp.303 A patient who was later found to be DPD-deficient developed severe neurotoxicity and remained in a comatose state for 4 days after bolus 5-FU/LV therapy; dramatic improvement in the neurologic status occurred after CI of dThd at 8 g/m2 per day.305

An uncommon complication of 5-FU and levamisole therapy is cerebral demyelination reminiscent of multifocal leukoencephalopathy.306, 307, 307a The symptoms occur after several months of adjuvant therapy, and include a decline in mental status, ataxia, and loss of consciousness. MRI scans with gadolinium enhancement show prominent multifocal-enhancing white matter lesions, and cerebral biopsy show morphologic features of an active, demyelinating disease. Myelin loss is associated with numerous dispersed and vasocentric macrophages, sparing of axons, and perivascular lymphocytic inflammation. Three patients improved after cessation of therapy and a short course of corticosteroids, but recovery was incomplete in two other patients. Because a similar phenomenon has not been reported in adjuvant studies involving 5-FU and LV or single-agent levamisole, the leukoencephalopathy may be unique to the combination.

A role for 5-FU catabolites is suggested by prolonged accumulation of [3H]FBAL as noted in brain tissue of rats.236 In a canine model, 5-FU administration with osmotic blood-brain barrier disruption produces neurotoxicity accompanied by foci of hemorrhagic necrosis and edema in brain tissue.308 The administration of eniluracil, an inhibitor of DPD, protects dogs from neurotoxicity associated with a 72-hour CI of 5-FU.309 Cats receiving either orally administered 5-FU or direct instillation of FBAL into the left ventricle have similar neuropathologic changes, and FBAL is more toxic than fluoroacetic acid, a potential metabolite of FBAL.310 Because neurotoxicity is a prominent feature of 5-FU toxicity in DPD-deficient patients, who cannot produce catabolites, direct effects of the 5FU or its anabolites may contribute to neurotoxicity.

In rhesus monkeys, after a 10-mg intraventricular dose, 5-FU disappears from ventricular CSF in a monoexponential fashion with a t1/2 of 51 minutes.311 The peak ventricular 5-FU concentration is 10 to 15 mmol/L, and the AUC is >18 mmol/L per hour, but without evident toxicity. After intralumbar administration, however, delayed onset of bilateral hindlimb paralysis was seen. Necropsy revealed abnormalities ranging from demyelination of the lumbar and sacral cords to severe necrosis of the ventral horn of the sacral spinal cord, and provides further evidence of direct 5-FU neurotoxicity. In vitro, FdUrd is more toxic to glial cancer cells than is 5-FU, but it is far less toxic to cultured neurons than 5-FU.312

Cardiotoxicity

5-FU therapy may be complicated by cardiac toxicity characterized by chest pain, arrhythmia, and changes in electrocardiograms (ECGs) with bolus and infusional schedules.313, 314, 315, 316, 317 Chest pain generally occurs in temporal association with 5-FU administration. The chest discomfort is often accompanied by ECG and serum enzyme changes indicative of myocardial ischemia. Some of these episodes have occurred in patients with a prior history of chest irradiation or cardiac disease, but coronary angiography performed subsequently showed no evidence of atherosclerotic disease, suggesting that coronary vasospasm might be involved. Cardiac shock and sudden death have also been reported. In a prospective multicenter cohort study of 483 patients receiving CI 5-FU, the incidence of suspected or documented cardiotoxic events was 1.9%; preexisting cardiac disease appeared to be a risk factor.315 There is no unequivocally effective prophylaxis or treatment for this syndrome. Once 5-FU administration is discontinued, symptoms are usually reversible, although fatal events have been described. There is a high risk of recurrent cardiac symptoms when patients are reexposed to this drug; therefore, it seems prudent to discontinue 5FU.

The pathophysiology of fluorouracil-associated cardiac adverse events is controversial. [3H]FBAL accumulates in cardiac tissue of rats for up to 8 days after a single dose.236 Fluoroacetic acid, a known cardiotoxic poison, was detected by fluorine-19 MRI in the perfusates of isolated rat hearts.318 Impurities such as fluoroacetaldehyde (which is metabolized into fluoroacetate) have been detected in the commercial formulation, and may result from degradation of 5-FU in the basic medium used to dissolve the drug.318a Fluoroacetate was detected in the urine of 15 patients treated with CI 5-FU, 6 of whom developed signs or symptoms of cardiac toxicity.318a Two patients who developed 5-FU-associated cardiac toxicity had high venous levels of endothelin-1, a potent naturally occurring vasoconstrictor, but whether this is cause or effect is unclear.319

Concentration-dependent vasoconstriction of smooth muscle in aortic rings freshly isolated from rabbits occurred in 23% and 54% of rings within minutes of exposure to 70 and 700 µmol/L 5-FU, but not with FdUrd.320 Pretreatment with inhibitors of protein kinase C reduced 5-FU-induced vasoconstriction, whereas protein kinase C activators increased it. Nitroglycerin abolished 5-FU-associated vasoconstriction in vitro.

Ocular Toxicity

5-FU may cause significant ocular toxicity, such as ocular irritation, tearing, epiphora, blepharitis, conjunctivitis, keratitis, eyelid dermatitis, cicatricial ectropion, tear duct stenosis, punctal-canalicular stenosis, and blurred vision.321, 322 Excessive lacrimation is the most frequent ocular symptom, but ocular pruritus and burning also occur. Conjunctivitis is reversible with discontinuation of 5-FU early in the patient's course, but progression of the inflammatory response may require surgical correction of dacryostenosis and ectropion. In a randomized, crossover trial in 62 patients with 5-FU-associated ocular toxicity, ocular ice pack therapy lessened 5-FU-induced ocular toxicity to a clinically moderate degree.323 Ocular toxicity often improves with dose reduction. Early ophthalmologic evaluation should be considered to avoid potentially permanent damage from fibrosis.

Pulmonary Toxicity with Systemic FdUrd

Three patients with renal cell cancer receiving FdUrd as a 14-day CI every 4 weeks developed nonproductive cough, dyspnea, and fever after a median of 15 months of therapy.324 Chest radiographs showed interstitial disease; pulmonary function tests revealed a restrictive pattern. Lung biopsies showed interstitial inflammation. The patients improved after discontinuation of FdUrd and institution of oral prednisone therapy, but required maintenance low-dose steroids to preserve their pulmonary function. One patient rechallenged with intravenous FdUrd developed recurrent symptoms. Pulmonary toxicity has has not been reported with single-agent 5-FU.

Toxicity of Hepatic Arterial Infusion

HAI of 5-FU or FdUrd is often used in patients with liver-only metastases to provide high local drug concentrations. Systemic toxicities are usually dose-limiting with HAI of 5-FU, presumably because more drug reaches the systemic circulation, and include oral mucositis, nausea, vomiting, and diarrhea.277, 278 Chemical hepatitis is usually mild. A strategy to limit systemic toxicity is to use continuous low-dose HAI of 5-FU.325

In contrast, systemic toxicities are uncommon with FdUrd, whereas hepatic toxicity is dose-limiting.280, 281, 283, 326, 327, 328, 329 Peptic ulcers, gastritis, and duodenitis occurred in up to 25% of patients in older studies, but the incidence has been substantially reduced with improved surgical technique (ligation of distal vessels that supply the superior border of the distal stomach and proximal duodenum, verification of catheter position). Chemical hepatitis, evidenced by elevations of alkaline phosphatase, transaminases, and bilirubin occurs commonly with HAI FdUrd. Cholestatic jaundice is a serious complication of HAI FdUrd, may progress to biliary sclerosis, and is believed to result from perfusion of the blood supply of the gallbladder and upper bile duct, via the hepatic artery. In more severely affected patients, cholangiograms reveal characteristic radiographic changes: narrowing of the common hepatic duct and the lobar ducts, varying degrees of intrahepatic ductal stricture, and sparing of the common bile duct. Liver biopsy reveals canalicular cholestasis and focal pericholangitis. The hepatocytes appear normal, although reactive changes (hyperplasia, intracellular bile staining, and small clusters of neutrophils in association with aggregates of Kupffer's cells) are present. Some patients require cholecystectomy for acalculous cholecystitis; at surgery, the gallbladder appears shrunken, hypovascular, and densely fibrotic. The onset of biliary sclerosis can be delayed by decreasing the initial dose (median time to toxicity at 0.2 or 0.3 mg/kg per day is five or three cycles). Although FdUrd may be reinstituted at a lower dose after normalization of liver enzymes, most patients became progressively intolerant. The clinical picture may not improve after interruption of therapy.

Patients receiving HAI FdUrd should have careful monitoring of the liver enzymes; therapy should be interrupted if elevations of alkaline phosphatase or transaminases occur. Imaging studies should rule out tumor progression. A randomized trial comparing HAI of FdUrd (0.3 mg/kg per day for 14 of 28 days) with or without dexamethasone (20 mg total) showed the incidence of patients experiencing a more than a twofold increase in bilirubin was decreased from 30 to 9%.327 The incidence of biliary sclerosis, 12%, seemed to be higher when low-dose LV was given concurrently with HAI FdUrd.328 In a subsequent phase 2 study in which dexamethasone, 20 mg total dose, was added to FdUrd (0.30 mg/kg per day) and LV (15 mg/m2 per day) as a 14-day HAI, the incidence of biliary sclerosis was only 3%.329

Catheter-related complications include arterial thrombosis, hemorrhage or infection at the arterial puncture site, and slippage of the catheter into the arterial supply of the duodenum or stomach, with necrosis of the intestinal epithelium, hemorrhage, and perforation. The occurrence of epigastric pain or vomiting should alert the clinician to promptly reassess the catheter position. In some patients, HAI may be impossible because of difficulties in catheter placement, thrombosis of the portal vein, or variations in vascular anatomy.

Age and Gender as Prognostic Factors for 5-Fluorouracil Clinical Toxicity

A number of clinical studies have reported significantly greater clinical toxicity in female and older patients treated with 5-FU-based therapy.330, 331, 332, 333,334, 335 Among 334 patients treated in a trial comparing monthly bolus 5-FU with weekly bolus 5-FU and LV, the incidence of clinical toxicities of grade 3 to 4 severity was significantly higher in patients older than 70 years compared with younger patients, and in women compared with men.330 Among 212 patients who received a monthly schedule of 5-FU and low-dose LV, the incidence of grade 3 to 4 leukopenia (21% versus 32% versus 40%) and mucositis (11% versus 26% versus 36%) increased with advancing age (<60 years, 60 to 69 years, and >70 years). Women had a higher incidence of grade 3 to 4 leukopenia than did men (39% versus 23%).331 Women experienced more severe neutropenia, diarrhea, and stomatitis than men in an adjuvant rectal study comparing two cycles of either 5-FU, 5-FU and LV, 5-FU and levamisole, or 5-FU, LV, and levamisole before and after chemoradiation with 5-FU±LV.332 The Meta-Analysis Group in Cancer, using individual data from six randomized trials comparing infusional with bolus 5-FU, found that female patients, older patients, and those with poorer performance had a significantly higher risk of diarrhea, mucositis, nausea, and vomiting.333 Hand-foot syndrome was 2.6-fold more common in patients receiving infusional 5-FU (34% versus 13%, P < .0001); female patients and older patients also had a higher risk of hand-foot syndrome. Grade 3 to 4 hematologic toxicity, mainly neutropenia, was sevenfold more common with bolus 5-FU therapy (31% versus 4%, P < .0001); poor performance status was a significant prognostic factor for serious hematologic toxicity.

The possible influence of age and gender on 5-FU clearance and DPD activity (in PBMCs or liver tissue) has yielded inconsistent results in different studies.208,238, 239, 240, 336, 337, 338 Even in trials that report a difference according to gender, there is considerable overlap in the values between men and women, and the correlation between either age and gender and 5-FU clearance or DPD activity is not tight. Other factors may also account for the increased toxicity in female and older patients. Age-related physiologic changes in the liver and kidneys, perhaps involving organ mass and function, and alterations in regional blood flow might account for the reduced elimination of metabolized drugs, such as 5-FU, in the older population.339

Because of the reports of increased clinical toxicity, it seems prudent to closely monitor blood counts and symptoms in older and female patients during 5-FU-based therapy with appropriate dose adjustments. It is not currently recommended that the dose of 5-FU be lowered a priori in these patient subsets.

Randomized Trials Comparing Various Fluoropyrimidine Routes and Schedules

A series of meta-analyses has been performed by the Advanced Colorectal Cancer Meta-Analysis Group (Table 7-10).340, 341, 342, 343, 344 CI is superior to intravenous bolus 5-FU when given as a single agent; hand-foot syndrome is significantly more common with CI (34% versus 13%), and hematologic toxicity is much less frequent (4% versus 31%).342 HAI of 5-FU or FdUrd consistently produced higher response rates in patients with liver metastases compared with systemic infusion of 5-FU or FdUrd.343 Improvement in survival was significant only when considering data from two trials in which patients randomized to the control arm may have remained untreated. The rationale for the use of LV and MTX as modulators of 5-FU is addressed subsequently.

PROTECTION STRATEGIES

A number of approaches have been explored in an effort to ameliorate toxic reactions of 5-FU in the experimental host (Table 7-11).

Allopurinol

Allopurinol is converted to oxypurinol ribonucleotide, which inhibits orotidylate decarboxylase and causes a buildup in the pools of orotidylate and orotic acid (orotate), which blocks 5-FU activation by OPRT.7, 9 Because host tissues, but not all tumors, may depend on this activation pathway, coadministration of allopurinol diminishes host toxicity in some in vivo models. Antitumor responses are maintained in tumors that use Urd phosphorylase and Urd kinase to activate 5-FU to FUMP. Patients receiving CI 5-FU were able to tolerate twice the daily dose of 5-FU if given with allopurinol (2.2 versus 1.1 g/m2 per day).345The granulocyte nadir in patients who received a single dose of 5-FU 1,200 mg/m2 alone the first cycle (mean, 577 per µL) was 75% lower than that observed during the second cycle, in which allopurinol was added to 5-FU. Allopurinol appeared to increase the maximally tolerated dose of 5-FU by 1.5- fold.301Reduction of 5-FU toxicity by allopurinol has not been consistent. High-dose allopurinol given with bolus 5-FU on biweekly and daily-for-5-days schedules was associated with unacceptable neurotoxicity.346, 347 A randomized trial showed no protection afforded by allopurinol mouthwash from 5-FU-associated mucositis.289 Allopurinol might potentially interfere with 5-FU activation in tumors that rely on the OPRT pathway. In a rat model, allopurinol decreased FdUMP formation in colon carcinoma tissue by less than twofold, but did not affect FdUMP levels in liver tissue.176

TABLE 7.10 META-ANALYSES COMPARING ROUTES AND SCHEDULES OF 5-FLUOROPYRIMIDINE THERAPY

Reference

Comparison

No. of Patients

% Responding (P value)

Median Survival (months) (P value)

340

Bolus 5-FU
Bolus 5-FU + leucovorin

578
803

11.1
22.5 (<1 × 10)–7

11
11.5

341

5-FU
5-FU + methotrexate

570
608

10
19 (<.0001)

9.1
10.7 (.024)

343

IV 5-FU or FdUrd or supportive care

655 (391 got 5-FU or FdUrd)

14

11 (12 months + chemotherapy)14

HAI FdUrd

654

41 (< 1 × 10)–10

16 (.0009 vs. all controls) (0.14 vs. + IV chemotherapy)

342

Bolus 5-FU
CI 5-FU

551
552

13.6
22.5 (.0002)

11.3
12.1 (.04)

By consuming PRPP, purine bases (e.g., hypoxanthine and adenine) can reduce 5-FU toxicity in some cell lines that use the OPRT pathway. Coadministration of hypoxanthine or adenine with 5-FU promptly reduced PRPP levels to less than 20% of baseline and reversed 5-FU toxicity in L5178Y cells.348, 349 There is currently no clinical role for allopurinol or purine bases as modulators of 5-FU.

Uridine Rescue

Several preclinical studies demonstrate a selective rescue of healthy tissue from 5-FU toxicity by delayed Urd.350, 351, 352, 353, 354 Delayed administration of pharmacologic doses of Urd (800 mg/kg every 2 hours for three doses followed 18 hours later by four doses every 2 hours) in tumor-bearing mice reduces 5-FU toxicity to the host without affecting its antitumor activity.350 Urd administration expands UTP pools and increases the clearance of [3H]5-FU from RNA and DNA in tumor and healthy tissues.351 Delayed administration of Urd doubles the tolerated dose of 5-FU given intraperitoneally once weekly, increases [3H]5-FU incorporation into RNA by 2.2-fold and improves experimental antitumor effect.354

TABLE 7.11 STRATEGIES TO REDUCE THE TOXICITY OF 5-FLUOROURACIL (5-FU) IN HEALTHY TISSUES

Modulator

Evidence for Protection in Preclinical Models

Reduction in Clinical Toxicity

Mechanism(s)

In vitro

In vivo

Allopurinol

Yes

Yes

Mixed results

Oxypurinol ribonucleotide inhibits orotate decarboxylase; orotate buildup inhibits 5-FU activation by orotate phosphoribosyl transferase; decreased leukopenia observed in some trials.

Purine bases

Yes

Yes

Unknown

Depletion of phosphoribosyl phosphate decreases 5-FU activation via orotate phosphoribosyl transferase.

Uridine rescue

Yes

Yes

Yes

Delayed administration of pharmacologic doses of uridine increases the clearance of 5-FU from RNA and DNA and allows faster recovery from RNA and DNA synthetic inhibition (host effect > tumor effect in tumors with limited capacity to salvage uridine).

Variable-rate CI “chronomodulation”

NA

Yes

Yes

In preclinical models, toxicity of 5-FU and 5-fluoro-2′-deoxyuridine influenced by time of administration; circadian-modulated CI regimens may reduce clinical toxicity of 5-fluoro-2′- deoxyuridine given systemically or by HAI.

Thymidine

Yes

No

No

Thymidine used in vitro to replete deoxythymidine triphosphate pools via thymidine kinase salvage, thus antagonizing DNA- directed toxicity of 5FU.

CI, continuous infusion; HAI, hepatic arterial infusion; NA, not applicable.

Delayed administration of Urd by CI (5 g/kg daily for 5 days subcutaneously) allows a threefold increase in 5-FU dose from 200 to 600 mg/kg and resulted in an improved therapeutic index in mice bearing B16 melanoma but not L1210 leukemia.352 An improved therapeutic index results from 5-FU and delayed Urd rescue in mice bearing either murine colon carcinoma 26 or 38, with less severe hematologic toxicity and more rapid recovery.353 The biochemical mechanism allowing selective Urd rescue of healthy tissues with retention of antitumor activity in these murine models is suspected to involve differences in Urd uptake and UTP-pool expansion, resulting in inhibition of further 5-FU-RNA formation and faster clearance of 5-FU from RNA. Tumors with limited capacity to salvage Urd represent the most logical model for selective protection.

In clinical trials, doses up to Urd 12 g/m2 given over 1 hour intravenously were well tolerated and increased plasma Urd above 100 µmol/L for up to 8 hours. However, delayed administration of 5 to 6 g/m2 24 and 48 hours after bolus 5-FU did not prevent myelosuppression.355 CI of Urd produced dose-limiting febrile reactions.356 An intermittent schedule of 3 g/m2 over 3 hours, alternating with a 3-hour rest, over 72 hours was tolerable and resulted in peak and trough Urd levels in the 1 mmol/L and 100 to 300 µmol/L range, respectively.357 This intermittent Urd schedule ameliorated leukopenia, but did not affect platelet toxicity.357 Phlebitis of peripheral veins necessitates administration of Urd via a central venous catheter.

Preclinical studies suggested that adequate plasma levels of Urd could be achieved by oral adminstration.358, 359 However, clinical studies suggested that the bioavailability of oral Urd indicated was only 6 to 10% for single-dose levels of 8 to 12 g/m2.360

Delayed administration of uridine as a modulator of 5-FU toxicity is currently being explored with triacetyluridine, an investigational Urd ester prodrug with high bioavailability. It remains to be resolved whether protection might be afforded to tumors with high levels of Urd kinase, and whether the timing of Urd rescue after 5-FU therapy might affect the rescue of healthy tissues versus tumor tissue.

Circadian-Dependent Toxicity of Fluoropyrimidines

In preclinical models, the time of administration of 5-FU and FdUrd influences host toxicity.361, 362, 363, 364, 365 In mice, drug administration during the active phase produces greater hematologic toxicity, whereas treatment during the rest phase results in longer tumor growth delay and smaller tumor volume in a 5-FU-sensitive tumor.362 The toxicity of an intraperitoneal bolus dose of FdUrd (1,200 mg/kg) administered at six different times was compared in rats. FdUrd was not lethal when administered at 12:00 PM (midpoint, resting phase), but killed 40% of rats when given at 4:00 AM (late, active phase).365

These observations may be explained by circadian-dependent changes in the rates of DNA and RNA synthesis and activities of enzymes such as dThd kinase and DPD, which are involved with 5-FU anabolism and catabolism.366, 367, 368, 369 Several studies in humans have also shown that DNA synthesis in bone marrow and intestinal mucosa follows a circadian pattern. The highest DNA synthetic rate in bone marrow occurs during the waking hours, with lowest DNA synthesis during the sleep span (12:00 AM to 4:00 AM).370, 371 5-FU Cp during fixed-rate CI may vary in a diurnal fashion, and an inverse correlation was reported between 5-FU plasma levels and DPD activity.205, 207 Other studies that monitored either 5-FU Cp or DPD activity on several occasions, however, reported marked interindividual and intraindividual variations.372, 373, 374, 375 It is possible to impose a circadian profile on 5-FU pharmacokinetics through the use of programmable infusion pumps (Fig. 7-6).373, 374

Several variable-rate infusion schedules have been explored in an effort to minimize host activity. When FdUrd was administered as either a fixed-rate infusion (0.15 mg/kg per day for 14 days) or a variable-rate infusion (15% of the total dose from 9:00 AM to 3:00 PM, 68% from 3:00 PM to 9:00 PM, 15% from 9:00 PM to 3:00 AM, 2% from 3:00 AM to 9:00 AM), the incidence and severity of diarrhea was significantly lower with the variable-rate infusion program.375Patients tolerated an average of 1.5- fold more FdUrd with minimal toxicity with variable-rate infusion.376 Variable-rate HAI of FdUrd appears to be less toxic than does fixed-rate infusion.377

Levi et al.378 developed a regimen involving a CI of 5-FU and LV (600 and 300 mg/m2 per day for 5 days) given between 10:15 PM and 9:45 AM, with peak delivery at 4:00 AM, and oxaliplatin (20 mg/m2 per day) given between 10:15 AM and 9:45 PM, with a peak at 4:00 PM, repeated every 21 days. A randomized trial comparing this regimen versus fixed-rate infusion of all three drugs concurrently over 24 hours daily in patients with metastatic colorectal cancer indicated a lower incidence of severe mucositis (18% versus 89%), higher median tolerated 5-FU dose (700 versus 500 mg/m2), and higher response rate (53% versus 32%) in favor of chronotherapy.378 A second trial confirmed these results.379 A randomized trial comparing chronomodulated 5-FU/LV (750/300 mg/m2 daily for 4 days over 11.5 hours with a peak at 4:00 AM) and oxaliplatin (25 mg/m2 daily for 4 days over 11.5 hours with a peak at 5:00 PM) with oxaliplatin 100 mg/m2 over 2 hours on day 1 with LV 250 mg/m2 intravenous over 2 hours followed by 5-FU 1500 mg/m2 intravenous over 22 hours on days 1 and 2, with both regimens repeated every 14 days demonstrated comparable efficacy.380

It is not clear whether the reduction in clinical toxicity with the 4-day chronomodulated regimen is the results of having an intermittent 11.5-hour exposure to drug with a 12.5-hour drug-free interval each day as opposed to specific timing of peak drug infusion. Other investigators have recommended a variable-rate schedule in which two-thirds of the total daily dose of 5-FU would be administered during the evening hours.381 Why different times for peak drug infusion are recommended in different trials is not clear. A general finding in the clinical trials evaluating variable-rate infusion schedules is that higher doses of 5-FU or FdUrd are tolerated, with reduced host toxicity.

Figure 7.6 5-Fluorouracil (5-FU) plasma levels during fixed and variable-rate infusion. 5-FU plasma levels were measured at 3-hour intervals over the second day of a 72-hour infusion of 5-FU 1,750 mg/m2 per day using either constant-rate infusion (top, n = 29) or a programmable infusion pump that delivered a smooth continuous sinusoidal variable infusion rate with a peak at 4 AM and a trough rate of 0 at 4 PM (bottom, n = 10). The data are shown as the mean and the standard deviation. (Reproduced with permission from the European Society for Medical Oncology from Grem, et al. Phosphonacetyl-L-aspartate and calcium leucovorin modulation of fluorouracil administered by constant rate and circadian pattern of infusion over 72-hours in metastatic gastrointestinal adenocarcinoma. Ann Oncol 2002;12:1581–1587)

Several key issues are unresolved. Do the major enzymes involved in 5-FU anabolism and catabolism in tumor tissue display circadian variation, and, if so, does the pattern differ from that of the healthy tissues? If the pattern of enzyme activity in tumor tissues parallels that of healthy tissues, then drug administration at a time intended to reduce host toxicity also may lead to decreased activation in tumor. Considering the clinical activity of the variable-rate infusion regimens, this may not be a major concern. However, given the diversity of human genetics, varying lifestyles with different sleep and wake cycles, geographic and seasonal changes that influence the duration of sunlight, the possible influences of other drugs, hormones, feeding and fasting, and rate of cell proliferation on circadian rhythms, the wide interpatient and intrapatient variability in diurnal profiles of 5-FU-plasma levels and DPD activity is perhaps to be expected.

BIOCHEMICAL STRATEGIES TO INCREASE THE CYTOTOXICITY OF 5-FLUOROURACIL

A number of important interactions have been demonstrated between 5-FU and other antineoplastic drugs or normal metabolites in experimental and clinical investigations (Table 7-12). These strategies include attempts to increase the conversion of 5-FU to its active metabolites, modulate its binding to TS, increase its incorporation into RNA or DNA, decrease the competing pools of normal nucleotides by blocking the de novo and salvage pathways of pyrimidine synthesis, decrease the catabolism of 5-FU and its metabolites, and combine 5-FU with other agents with complementary mechanisms of cytotoxicity. Several of these strategies have yielded convincing evidence of clinical benefit, whereas others have failed to improve the therapeutic index.

Sequential Methotrexate-Fluorouracil

5-FU and MTX both inhibit the synthesis of dTMP and dTTP by either binding of FdUMP to TS or depletion of intracellular reduced folates and the generation of toxic polyglutamates (Fig. 7-7). Several biochemical interactions are possible.382 The reduced folate 5,10-CH FH, which is required for binding of FdUMP to TS, is oxidized to FH2 in the TS reaction and cannot be resynthesized in the presence of MTX. Pretreatment of cells with MTX depletes 5,10-CH FH, which could interfere with FdUMP binding to TS.26 Because depletion of reduced folates by MTX is only partial, however, this may be insufficient to affect the binding of FdUMP to TS. FH2 polyglutamates, which accumulate in the presence of antifolates, may substitute for 5,10-CH FH4 in the ternary complex with FdUMP and TS.25 MTX pretreatment also results in accumulation of dUMP, which may compete with FdUMP for binding to TS.

MTX pretreatment augments FUTP formation. Above threshold concentrations of 0.1 to 1.0 µmol/L, MTX inhibits de novo purine synthesis, thus expanding the intracellular pool of PRPP. PRPP is then available for the conversion of 5-FU to FUMP by OPRT; FUTP formation, 5-FU-RNA incorporation, and cytotoxicity are thereby increased, leading to cytotoxic synergism when MTX precedes 5-FU in L120 cells.9 With cultured human cancer cells tumors, longer periods of incubation with MTX are required to produce expansion of the PPRP pools. Enhancement of 5-FU toxicity in some human cancer cells is greatest when exposure to ≥1 µmol/L MTX is maintained for 24 hours before 5-FU.11, 383, 384 In MCF-7 breast cancer cells, in contrast, pretreatment with MTX did not enhance 5-FU activation.384 The synergistic cytotoxicity of sequential MTX 5-FU in vitro requires medium containing serum with low concentrations of hypoxanthine and dThd because the addition of physiologic concentrations of hypoxanthine (1 to 10 µmol/L) and dThd (0.5 µmol/L) to dialyzed serum antagonizes the synergistic effects of sequential exposure.385 Several in vivo models have shown improved antitumor activity when MTX is given 22 to 24 hours before bolus 5-FU, along with increased formation of 5-FU ribonucleotides and 5-FU-RNA incorporation.386, 387

The reverse sequence of drug administration (5-FU followed by MTX) produces the least favorable antitumor effects in cell culture and in vivo.9, 382, 387, 388,389 The sequence-dependent antagonism is a consequence of antagonism of the antipurine effects of MTX by 5-FU. The antipurine action of MTX is believed to result from two factors: partial depletion of 10-formyl FH and buildup of FH, an inhibitor of de novo purine synthesis. Ongoing dTMP synthesis is required to deplete the cellular reduced-folate pool. Pretreatment with 5-FU inhibits TS, thereby blocking the conversion of reduced folates to FH. The reduced-folate pool is thus spared for purine synthesis, and the FH2 pool does not expand.389 When MTX precedes 5-FU, ongoing dTMP synthesis leads to depletion of reduced folates and accumulation of FH2 pools, thus allowing blockade of purine biosynthesis.

In summary, despite potential interference with the formation of the ternary complex involving FdUMP, TS, and reduced folate, experimental sequences using MTX before 5-FU have produced more favorable results than have regimens using 5-FU first, presumably through enhancement of the RNA-directed toxicities of 5-FU. The dosage of MTX and the interval before 5-FU administration must be sufficient to allow the biochemical effects of MTX to become established. Pharmacodynamic studies suggest that PRPP levels are significantly increased 24 hours after MTX.390 One randomized trial in colorectal cancer demonstrated that a 24-hour interval between MTX and 5-FU was superior to a 1-hour interval.391 In contrast, a trial involving MTX, 5-FU, and LV in patients with head and neck cancer showed no advantage for an 18-hour interval compared with concurrent administration in terms of response rate, but host toxicity was greater with sequential MTX 5-FU and LV.392 These results suggest that the ability of MTX to modulate 5-FU toxicity and the optimal time of administration may depend on the tissue type. A meta-analysis of eight randomized trials comparing MTX modulation of 5-FU with bolus 5-FU alone in patients with metastatic colorectal cancer documents a doubling of the response rate with MTX modulation (Table 7-10).341

TABLE 7.12 STRATEGIES TO ENHANCE THE CYTOTOXICITY OF 5-FLUOROURACIL (5-FU) IN CANCER CELLS

Modulator

Evidence for Increased Cytotoxicity in Preclinical Models

Enhancement of Clinical Activity

Putative Mechanism(s)

In vitro

In vivo

Methotrexate

Yes

Yes

Yes: phase 3 trials

Sequential methotrexate 5-FU inhibits de novo purine synthesis, causing expansion of phosphoribosyl phosphate pools, increased formation of FUTP, and increased incorporation of FUTP into RNA.

Leucovorin

Yes

Yes

Yes: phase 3 trials

Expansion of 5,10-CH FH monoglutamate and polyglutamate pools increases the stability of the reduced folate-fluorodeoxyuridylate-TS ternary complex; enhances DNA-directed effects of 5-FU.

Thymidine

Yes

Yes

No: phase 1, 2 trials

Thymidine antagonizes the DNA-directed effects of 5-FU, but pharmacologic doses may increase 5-FU anabolism to FUTP and increase FUTP-RNA incorporation; thymidine and thymine competitively decrease 5-FU catabolism by dihydropyrimidine dehydrogenase, markedly prolonging the t1/2 of 5-FU and increasing toxicity to the host.

N-(phospho-noacety l)-l-aspartic acid, brequinar

Yes

Yes

N-(phosphono-acetyl)-l-aspartic acid: No: randomized phase II and III trials

Inhibition of de novo pyrimidine synthesis leads to depletion of uridine triphosphate and cytidine triphosphate pools, which compete with FUTP for RNA incorporation; less feedback inhibition of uridine kinase; decreased uridine triphosphate and cytidine triphosphate pools in turn result in decreased formation of deoxyuridine monophosphate and deoxycytidine triphosphate; increased phosphoribosyl phosphate pools and decreased production of orotic acid favor formation of fluorouridine monophosphate; increased FUTP incorporation into RNA; may increase RNA and DNA-directed toxicities of 5-FU.

IFN α,β,γ

Yes

Yes

IFN-α: No: phase III trials

Mechanism of interaction may differ in various cancer cell lines; IFN-α may increase fluorodeoxyuridylate formation, enhance DNA-damage, and potentiate natural killer cell–mediated cytotoxicity; IFN-γ may abrogate acute increase in TS content during 5-FU exposure, thus extending the extent and duration of TS inhibition; in vivo, IFN-α may affect 5-FU clearance in a dose and schedule-dependent manner in some individuals. 146 FUTP, fluorouridine triphosphate; IFN, interferon; TS, thymidylate synthase.

Cisplatin and analogs

Yes

Yes

Cisplatin: Yes: phase III trials, squamous cell cancers
Oxaliplatin: Yes: phase III trials in colorectal cancer

Cisplatin may indirectly increase the FH4 and 5,10-CH2 FH4 pools; 5-FU may interfere with the repair of cisplatin- associated DNA damage. Oxaliplatin is active against DNA mismatch repair deficient cancer cells; clinical evidence of synergy between oxaliplatin and 5-FU in colorectal cancer; underlying mechanism uncertain.

Ionizing radiation

Yes

Yes

Yes: phase III trials: various squamous cell cancers and rectal cancer

Augmentation of DNA directed cytotoxicity.

When higher-than-standard doses of MTX are used, LV rescue is used to protect the patient, which may contribute to the improved activity of MTX, 5-FU, and LV regimens. The strategy of LV rescue is based on the assumption that delayed administration of LV will be more likely to rescue healthy tissues than tumor tissues, although the potential for tumor protection remains a concern. Substitution of the lipophilic-antifolate trimetrexate for MTX in regimens involving sequential antifolate 5-FU with LV rescue has been recommended because trimetrexate and LV do not compete for transport or polyglutamation. Sequence-dependent synergism is seen with trimetrexate given before 5-FU in preclinical models.392, 393 Despite promising results in phase 2 studies in colorectal cancer with a regimen of trimetrexate, 5-FU and LV, no benefit of this three-drug combination was seen when compared with 5-FU and LV alone in two randomized clinical trials in previously untreated colorectal cancer.395

Figure 7.7 Interaction between 5-fluorouracil (5-FU) and methotrexate (MTX). A. MTX preceding 5-FU. DHFR, dihydrofolate reductase; dTMP, thymidine 5′-monophosphate; dTTP, thymidine 5′-triphosphate; dUMP, deoxyuridine monophosphate; FdUMP, fluorodeoxyuridylate; PP, pyrophosphate; PRPP, phosphoribosyl phosphate.

MODULATION OF FLUORINATED PYRIMIDINES BY FOLINIC ACID

The ability to form and maintain a stable ternary complex is a critical determinant of sensitivity to 5-FU and FdUrd. The concentration of reduced folate in equilibrium with the ternary complex inversely correlates with its rate of dissociation.32, 396, 397, 398, 399 High levels of intracellular reduced folates are, therefore, necessary for the optimal binding and inhibition of TS by FdUMP. Enhanced inhibition of TS over a sustained period results in further depletion of dTTP pools, greater inhibition of DNA synthesis, increased DNA damage, and enhanced cytotoxicity. The endogenous reduced-folate levels are insufficient to promote maximal inhibition of TS in many tumors; in 5-FU-sensitive tumors, in contrast, maximal FdUMP binding into the ternary complex occurs without exogenous LV.397, 398, 399, 400, 401 LV (folinic acid, citrovorum factor, 5-formyltetrahydrofolate, 5-CHO-FH) has been used to expand intracellular reduced-folate pools and thereby permit maximal ternary-complex formation.162 LV increases the in vitro and in vivo cytotoxicity of 5-FU in many, but not all, cancer cell types in a concentration and time-dependent manner.27, 95, 96, 163, 164, 165, 186, 401, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411 LV concentrations below 1 µmol/L are insufficient to expand intracellular folate pools, and 10 µmol/L is often cited as the target concentration. As the duration of exposure increases, the concentration of LV required to optimally modulate total intracellular levels of 5,10-CH FH4 and enhance fluoropyrimidine toxicity decreases. With brief exposures to 5-FU or FdUrd, several preclinical studies suggest the importance of giving LV before or concurrently with the fluoropyrimidine to permit metabolism of LV to 5,10-CH FH4 polyglutamates, which are more effective in promoting ternary-complex formation.398, 402, 406, 503 Other investigators have argued that 5-FU should be given 30 to 40 minutes before LV to achieve peak concentrations of FdUMP and 5,10-CH FH4 simultaneously.399, 409 The optimal concentration of LV and time of administration relative to 5-FU exposure may vary depending on the tumor model used. In two different human cancer cell lines, maximum ternary complex formation occurred when 5-FU exposure was delayed for either 4 hours or 18 hours after exposure to LV; the time of peak folate-polyglutamate formation coincided with the time of peak TS complex formation and total TS protein in each cell line.403 The ability of LV to modulate cytotoxicity is also influenced by the duration of exposure to 5-FU.404

LV is chemically synthesized and consists of equal amounts of the diastereoisomers R- (or D-) and S- (or L-) 5-CHO-FH. The natural diastereoisomer is S-5-CHO- FH4, which must be metabolized to exert its modulatory effects on 5-FU (Fig. 7-8). After intravenous administration, S-5-CHO-FH is rapidly cleared from plasma by conversion to its metabolite S-5-methyl-tetrahydrofolate (5-CH-FH) and by urinary excretion. 5-CHO-FH and 5-CH-FH4 are transported across the cell membrane by a common saturable reduced-folate carrier and then undergo complex intracellular metabolism, including polyglutamation. An important determinant of sensitivity to LV modulation is variation in the intracellular metabolism of 5-CHO-FH4 and 5-CH-FH to 5,10-CH FH4 and its conversion to polyglutamates. In cell-free systems, 5,10-CH FH4 with a five-chain length polyglutamate binds more tightly to TS than the monoglutamate (apparent Km, 0.6 versus 23 µmol/L for Glu-5 and Glu-1, respectively) and is 40-fold more potent in promoting ternary-complex formation.24 The intracellular t1/2 increases as the number of glutamate residues increases. 5,10-CH FH4 with three or six glutamate residues was 18-fold and 200-fold more effective in stabilizing the ternary complex with TS purified from a human colon cancer than the monoglutamate form.407 The increase in total reduced-folate cofactor content is concentration-dependent; prolonged exposure is necessary to permit accumulation of the more potent longer chain length polyglutamates.163, 164, 165, 407, 410 Cell lines with impaired ability to transport, metabolize, and polyglutamate reduced folates are relatively insensitive to LV modulation of 5-FU toxicity in proportion to the severity of the metabolic defect in folate metabolism.161, 166, 410 Although exposure to higher doses of LV may not be necessary to promote optimal formation and stabilization of ternary complex in all cancer cell types, increasing the dosage and duration of LV exposure may sensitize certain tumors that are otherwise unaffected by low-dose or short-term exposure to LV. There appears to be no advantage to using the active stereoisomer compared with racemic LV.162, 163, 164, 412

Figure 7.8 Interconversions of reduced folates. ADP, adenosine 5′-diphosphate; 5-AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; ATP, adenosine 5′-triphosphate; dTMP, thymidine 5′-monophosphate; dUMP, deoxyuridine monophosphate; 5-FAICAR, 5-formamidoimidazole-4-carboxamide ribonucleotide; FdUMP, fluorodeoxyuridylate; MTX, methotrexate; NADP, nicotinamide adenine dinucleotide; NADPH, nicotinamide adenine dinucleotide phosphate;–, inhibition.

The combination of 5-FU and LV has been extensively tested in the clinic. Dose, route, and schedule of LV administration have varied. Pharmacokinetic studies have been performed for several LV regimens. With intravenous bolus injection of 50 mg racemic LV, plasma levels of bioactive reduced folates (S-5-CHO-FH and 5-CH-FH) remain above 1 µmol/L for 1 hour.413 When the intravenous bolus dose is increased to 200 mg/m2 and with a 2-hour infusion of 500 mg/m2 LV, peak plasma levels of bioactive reduced folates exceed 40 µmol/L.414, 415 With CI of 500 mg/m2 LV, the Css values of S-5-CHO-FH and 5-CH3-FH are 4 to 5 µmol/L each.416 Bioavailability studies after five different dosages ranging from 10 to 500 mg/m2 showed that LV absorption with oral dosing was saturable. Accumulation of several metabolites was greater after intravenous than oral administration, and peak plasma levels of FH and 5,10-CH-FH exceeded 2 µmol/L after an intravenous dose of 500 mg/m2.417 Host-mediated biotransformation of LV to the active metabolite occurs before tumor uptake, which may provide an added rationale for high-dose intravenous LV.

Numerous randomized phase 3 trials have evaluated the worth of LV-modulation of intravenous bolus 5-FU. The tolerated dose of 5-FU when given in combination with LV is lower than that for single-agent 5-FU, and increased GI epithelial toxicity is noted. A meta-analysis of nine randomized trials of 5-FU and LV compared with 5-FU alone in patients with advanced colorectal cancer indicated that 5-FU and LV therapy showed a highly significant benefit over single-agent 5-FU in terms of tumor response rate, although this did not translate into a survival advantage, but no apparent differences were noted between weekly and monthly (daily for 5 days) schedules in the meta-analysis of 5-FU and LV trials340 (Table 7-10). Randomized trials comparing the monthly 5-FU and low-dose LV with weekly 5-FU high-dose LV schedules in advanced colorectal cancer and as adjuvant therapy reveal similar efficacy, with different toxicity profiles.418

Interferon with 5-Fluorouracil

Numerous in vitro studies have demonstrated that interferons (INFs) α, β and γ may interact with 5-FU or FdUrd in a greater than additive fashion to produce cytotoxicity in a variety of human cancer cell lines.177, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430 The type of IFN that maximally enhances fluoropyrimidine cytotoxicity differs among cell lines. Because of the ability of dThd to rescue cells from the additive effects of IFN, enhancement of the DNA-directed actions of 5-FU is implicated.92, 108, 177, 181, 419, 424, 425 In several models, pretreatment with IFN for 24 to 48 hours followed by concurrent exposure to 5-FU and IFN produced optimal effects; other studies gave IFN concurrently with 5-FU for 24 to 72 hours. New protein synthesis appears to be a requirement for IFN-mediated augmentation of fluoropyrimidine toxicity in some studies.108, 422, 423, 426, 428 In some leukemia and colon cancer cell lines, IFN increases FdUMP formation and enhances TS inhibition, apparently as the result of an increase in the activities of dThd phosphorylase.420, 421, 422

In other models, enhancement of 5-FU cytotoxicity by IFN is noted in the absence of an effect on 5-FU metabolism or the extent of TS inhibition; the locus of interaction appears to be at the level of DNA damage.424, 425, 430 In H630 colon cancer cells, IFN-(abrogates the 5-FU-induced increase in TS content by interfering with TS-mRNA translation, leading to enhanced inhibition of TS.177, 181 In HT29 colon cancer cells, the combination of IFN-”

IFN-(and 5-FU led to more than additive cytotoxic effects.430 More profound dTTP depletion occurred with the IFNs combination compared to 5-FU alone; this was not caused by enhanced TS inhibition. The exaggerated dTTP depletion was accompanied by greater imbalance in the ratio of dATP to dTTP pools, and more pronounced inhibition of DNA synthesis and damage to nascent and parental DNA.430

The underlying basis for enhancement of 5-FU toxicity by IFN is variable, and the implicated mechanisms appear to depend on the specific cancer cell line or tumor model studied. In addition to biochemical and molecular mechanisms, immunomodulatory effects may be operative in vivo.426, 427

The potential impact of IFN-” on the activity of 5-FU plus or minus LV has been extensively evaluated in clinical trials using a variety of dosages and schedules. Although the response rates in phase 2 studies seemed much higher than expected with 5-FU or 5-FU and LV alone, meta-analysis of results from randomized trials in advanced colorectal cancer has failed to support a clinical benefit for IFN, although IFN definitely increases host toxicity, particularly with higher dosages344 (Table 7-10).

The increased toxicity with IFN-” and 5-FU alone or with LV suggests no selective preference for tumor over host tissues. The basis for the enhanced clinical toxicity may, in part, be explained by alterations in dThd phosphorylase expression in host tissues and by a pharmacologic interaction between IFN-” and 5-FU. Several investigators have reported a decrease in 5-FU clearance by IFN-”, perhaps because of interference with the ability of DPD to catabolize 5-FU, particularly with consecutive daily dosing of IFN and 5-FU.198, 431, 432, 433, 434 The correlation noted between higher 5-FU AUC or Css and increased GI toxicity in several of these studies suggests that the pharmacokinetic interaction of IFN-” with 5-FU contributes to the toxicity of the combination.199, 431, 434

Fluorouracil and Thymidine

Although dThd is known to reverse the cytotoxicity of low concentrations of 5-FU in vitro, high concentrations of 5-FU (above 10 µmol/L) may not be countered effectively by dThd in all cancer cells. Enhancement of 5-FU potency by pharmacologic doses of dThd has been observed in vivo.37, 38, 39 Thymine, produced from the catabolism of dThd by dThd phosphorylase, competes with 5-FU for degradation by DPD, thus prolonging the plasma t1/2 of 5-FU (Fig. 7-9). dThd is anabolized to dTMP via dThd kinase, which has several consequences. First, dThd can compete with FdUrd for dThd kinase, thereby decreasing FdUMP formation. Subsequent metabolism of dThd monophosphate to dTTP will expand the dTTP pools; dTTP, in turn, acts as a feedback inhibitor of dThd kinase and ribonucleotide reductase. Inhibition of the latter enzyme prevents FUDP conversion to FdUDP and consequently FdUMP, thus allowing enhanced FUTP formation and its incorporation into RNA. dThd acts as a donor of the deoxyribose moiety to promote the direct conversion of 5-FU to FdUrd by dThd phosphorylase; in some models, low concentrations of dThd may promote 5-FU incorporation into DNA.80 Pharmacologic concentrations of dThd are intended to increase the RNA-directed effects of 5-FU while negating the DNA-directed toxicity.37, 38, 39, 435

Clinical trials of the 5-FU and dThd combination confirmed enhanced 5-FU toxicity.254, 255, 256, 435, 436 CI of dThd 8 g/day produces a Css of approximately 1 µmol/L; when given with 5-FU doses of 370 to 555 mg/m2 daily for 5 days, severe myelosuppression and mucositis occurred.436 Pretreatment of patients with dThd (7.5 to 45 g) 1 hour before 5-FU produced severe thrombocytopenia, leukopenia, mucositis, and diarrhea.255 dThd markedly alters 5-FU plasma pharmacokinetics.254, 255, 256

Figure 7.9 Interaction between 5-fluorouracil (5-FU) and thymidine (dThd). DHFU, dihydrofluorouracil; dR-1-P, deoxyribose-1-phosphate; dTDP, thymidine 5′-diphosphate; dThd, thymidine; dTMP, thymidine 5′-monophosphate; dTTP, thymidine 5′-triphosphate; dUMP, deoxyuridine monophosphate; FdUDP, fluorodeoxyuridine diphosphate; FdUMP, fluorodeoxyuridylate; FdUrd, 5-fluoro-2′-deoxyuridine; FUDP, fluorouridine diphosphate; FUMP, fluorouridine monophosphate; FUrd, 5-fluorouridine; FUTP, fluorouridine triphosphate; Thy, thymine;–, inhibition.

 

The clinical results do not indicate a differential effect on tumor cells as opposed to the host. Severe hematologic, GI, and CNS toxicity has been a feature of virtually all 5-FU/dThd regimens irrespective of dose or schedule. The increase in clinical toxicity with the combination of dThd and 5-FU required a more than 50% 5-FU dose reduction from conventional regimens, with no improvement in antitumor activity.

5-Fluorouracil and Inhibitors of de Novo Pyrimidine Biosynthesis

A number of inhibitors of the de novo synthesis of pyrimidines have been evaluated as modulators of 5-FU, including pyrazofurin and 6-azauridine, inhibitors of orotidylate decarboxylase; acivicin, an inhibitor of carbamoylphosphate synthetase and CTP synthase; PALA, an inhibitor of aspartate carbamoyltransferase; and brequinar, an inhibitor of dihydroorotate dehydrogenase (Fig. 7-10).437, 438, 439, 440, 441, 442, 443, 444 Inhibition of specific steps in the de novo pathway by these compounds causes reductions in pyrimidine nucleotide pools and promotes use of preformed pyrimidines, such as 5-FU. Inhibitors of orotate decarboxylase also elevate intracellular levels of orotic acid, which competitively inhibits the conversion of 5-FU to FUMP by OPRT.

Figure 7.10 Sites of action of inhibitors of de novo pyrimidine biosynthesis. CPE-C, cyclopentenyl cytosine; CTP, cytidine triphosphate; OPRTase, orotate phosphoribosyl transferase; PALA, N-phosphonoacetyl-L-aspartic acid; PRPP, phosphoribosyl phosphate; R-1-P, ribose-1-phosphate; UDP, uridine diphosphate; UMP, uridine monophosphate; Ura, uracil; Urd, uridine; UTP, uridine triphosphate;–, inhibition.

PALA inhibits the second step in the de novo pathway of pyrimidine biosynthesis. PALA-mediated depletion of UTP and CTP results in diminished feedback inhibition of Urd-cytidine kinase activity, thereby favoring formation of FUMP through the salvage pathway and decreased competition with FUTP for RNA polymerase.445 Inhibition of the de novo pathway increases the availability of PRPP and decreases formation of orotic acid, thus favoring the formation of FUMP via OPRT. Depletion of pyrimidine nucleotide pools decreases dUMP formation through the ribonucleotide reductase pathway, with less competition with FdUMP for TS binding. Finally, decreased deoxycytidine triphosphate pools might enhance the DNA-directed toxicity of 5-FU. A number of these biochemical effects have been noted in preclinical in vitro and in vivo models, and a common feature of PALA modulation is increased 5-FU incorporation into RNA.

Preclinical studies showing enhancement of 5-FU activity with low-dose PALA led to clinical interest in exploring lower, biochemically active dosages of PALA given with full-dose 5-FU. Pharmacodynamic studies have led to different recommendations regarding the PALA dosage, depending on the biochemical indicator used. In a phase 1 study of PALA given 24 hours before intravenous bolus 5-FU, the biochemical effects of PALA were monitored using a surrogate healthy tissue end point: the effect on pyrazofurin- mediated urinary excretion of orotic acid and orotidine.446 Because PALA (250 mg/m2) was associated with a biochemical effect and allowed administration of full-dose bolus 5-FU, this dose was selected for subsequent clinical studies. Other studies that directly monitored ACTase activity suggested incomplete and transient inhibition by PALA 250 mg/m2.211, 447

Nonrandomized studies reported encouraging response rates in patients with colorectal cancer who were treated with PALA, 250 mg/m2, given 24 hours before a 24-hour CI of 5-FU 2,600 mg/m2 on a weekly schedule, but randomized trials failed to support a benefit of low-dose PALA.270, 272

Hydroxyurea

Hydroxyurea, an inhibitor of ribonucleotide reductase, has been combined with 5-FU by virtue of its ability to decrease dUMP formation. In mice bearing L1210 leukemia, the combination of hydroxyurea (100 mg/kg daily intraperitoneally) and FdUrd (75 mg/day intraperitoneally on days 1 to 5, 8, 11, and 14) resulted in significantly longer survival compared with either drug alone at optimal dosages.448 The combination of hydroxyurea and 5-FU has been explored clinically in the treatment of colorectal carcinoma, but several randomized trials have shown no apparent advantage.449, 450, 451

Combination with Purines and Pyrimidines

In cell lines that possess Urd phosphorylase, dThd phosphorylase, Urd kinase, and dThd kinase, simultaneous exposure of cancer cells to 5-FU and purines, such as inosine and deoxyinosine, or low concentrations (10 to 25 µmol/L) of pyrimidines, such as Urd and dUrd, may enhance 5-FU toxicity.73, 131, 452, 453,454, 455 In a human colon cancer cell line, the addition of 25 µmol/L dUrd to 5-FU enhanced FdUMP and dUMP formation, the extent of DNA damage, and cytotoxicity.73 Urd functions as a potent inhibitor of DPD (Ki [inhibition constant] 0.7 µmol/L for DPD isolated from hepatic tissue); at 10 µmol/L, Urd totally inhibits 5-FU catabolism by this enzyme,456 which highlight the importance of sequence of administration and the drug concentration to the outcome.

Healthy tissues can catabolize FdUrd by either Urd phosphorylase or dThd phosphorylase. Some malignant cells are known to be deficient in dThd phosphorylase while retaining Urd phosphorylase. In such tumors, the combination of FdUrd with an inhibitor of Urd phosphorylase is predicted to prevent its catabolism to 5-FU and increase anabolism to FdUMP. In contrast, host tissues that contain dThd and Urd phosphorylases would retain the ability to catabolize FdUrd, despite inhibition of the latter enzyme. This hypothesis has been confirmed with benzylacyclouridine (BAU), an inhibitor of Urd phosphorylase. BAU potentiates the cytotoxicity of FdUrd against human carcinoma cell lines in vitro and in vivo.458, 459 Delayed administration of BAU in combination with Urd reduces host toxicity from 5-FU; the combination of 5-FU and BAU, with or without Urd, was more effective than 5-FU alone against murine colon tumor 38 in vivo.459

5-Fluoropyrimidines as Biochemical Modulators of Other Halogenated Pyrimidines

The halogenated pyrimidines iododeoxyuridine (IdUrd) and bromodeoxyuridine (BdUrd) have been studied as radiosensitizers because their 5′-triphosphate metabolites are incorporated into DNA.460 IdUrd has cytotoxic properties as a single agent. Iododeoxyuridine triphosphate (IdUTP) and bromodeoxyuridine triphosphate (BrdUTP) compete with dTTP for incorporation into DNA. Iododeoxyuridine and bromodeoxyuridine monophosphate are substrates for TS; this interaction results in cleavage of the iodine or bromine from the carbon-5 position.461 Coadministration of an inhibitor of TS is expected to diminish the inactivation of iododeoxyuridine monophosphate and bromodeoxyuridine monophosphate, whereas depletion of dTTP should stimulate incorporation of IdUTP and BrdUTP into DNA. The success of this approach depends on duration of exposure and substrate competition for phosphorylation by dThd kinase. The activity of dThd kinase is dependent on feedback inhibitors, such as dTTP and IdUTP. 5-FU may be converted to FdUMP by alternate pathways not involving dThd kinase, thus avoiding potential competition with IdUrd or BdUrd. In several in vitro and in vivo models, 5-FU and FdUrd increase the DNA incorporation of IdUTP or BdUTP, resulting in increased cytotoxicity, radiosensitization, or both.462, 463, 464, 465, 466 In a human bladder cancer cell line, enhancement of IdUrd-DNA incorporation and cytotoxicity requires exposure times of 4 to 24 hours.463 In contrast, a concurrent 1-hour exposure to 3 µmol/L each of IdUrd and FdUrd is antagonistic, with decreased formation of FdUMP.466 In HT 29 colon cancer cells, the increased radiosensitization appears to result from decreased dTTP pools accompanied by increased incorporation of IdUrd into DNA, and cell-cycle redistribution with an accumulation of cells in the G1 and S phases.465

In early clinical trials, IdUrd was generally given by short intravenous infusions of 2 hours or less. The combination of FdUrd and IdUrd was associated with some tumor regressions, but toxicity was severe.467, 468, 469 Prolonged CI of 1,000 mg/m2 per day IdUrd for 14 days can be administered safely; at this dose, IdUrd Css of 3 µmol/L were achieved, and up to 11% substitution of dThd by IdUrd was observed in the DNA of peripheral granulocytes.470, 471, 472 Selective incorporation of IdUrd into DNA of hepatic tumor was seen compared with healthy liver when IdUrd was given by either systemic CI or HAI.473 IdUrd (200 to 675 mg/m2 per day) and FdUrd (0.6 to 3.5 mg/m2 per day, 15 to 78% of the single-agent highest tolerated dose) were subsequently combined as a concurrent 14-day CI.474 At a fixed dosage of IdUrd, increasing dosages of FdUrd did not appear to increase IdUrd Css or percent IdUrd substitution. With escalating doses of IdUrd and fixed doses of FdUrd, the Css for IdUrd rose proportionally, as did the percent IdUrd substitution, but no relevant enhancement of IdUrd incorporation into DNA by FdUrd was evident. Dose-limiting toxicities included thrombocytopenia, diarrhea, mucositis, and elevation of serum transaminases.474 The addition of LV 200 mg/m2 to a 14-day infusion of IdUrd reduced the highest tolerated dose to 400 mg/m2 (Css 0.7 µmol/L), but did not enhance IdUrd-DNA incorporation in peripheral blood granulocytes.475

A 14-day HAI of 5-FU 300 mg/day was studied with escalating doses of IdUrd given as a 3-hour HAI on days 8 to 14.476 With IdUrd 37 to 81 mg/m2 per day, the systemic peak plasma levels of IdUrd (0.2 to 0.8 µmol/L) and iodouracil (0.4 to 1.8 µmol/per L) increased. Although 5-FU was undetectable during infusion of 5-FU alone, Cp of 0.5 µmol/L were seen during infusion of IdUrd doses of 37 mg/m2 per day or more.476 Hepatic toxicity was dose-limiting; biliary sclerosis was documented in one patient. Although tumor regression occurred in some cases, the use of this regimen is limited by hepatic toxicity, reminiscent of that seen with HAI of FdUrd.

Combination with Nucleoside Transport Inhibitors

Human colon carcinomas possess high levels of the enzymes necessary for nucleoside salvage, and dThd salvage represents a potential mechanism of resistance to 5-FU or FdUrd. Dipyridamole, nitrobenzylthioinosine, and dilazep inhibit the uptake and efflux of nucleosides, such as dThd, FdUrd, and dUrd, in a dose-dependent manner.477, 478, 479 Because the effects of dipyridamole on nucleoside transport are rapidly reversible on drug removal, continuous exposure is necessary to modulate 5-FU toxicity. In HCT 116 colon cancer cells, dipyridamole and NBMPR increase FdUMP formation and the cytotoxicity of 5-FU.478, 479,480 Augmentation of 5-FU toxicity is concentration-dependent; free dipyridamole concentrations as low as 50 nmol/L modulated 5-FU toxicity, but optimal effects required 500 nmol/L.478 Increased FdUMP levels result in part from blockade of the efflux of FdUrd and other deoxyribonucleosides, such as dUrd, which serve as donors of dR-1-P.73, 479 Expansion of dUMP pools with 5-FU is accompanied by increased production of alkaline-labile sites in newly synthesized DNA.73 A direct correlation is noted between increased accumulation of dUTP and increased DNA fragility in cells treated with the antifolate TS inhibitor CB3717 and dipyridamole.74 The interaction between nucleoside transport inhibitors and 5-FU and FdUrd is complex, and more than one mechanism may exist.

The combination of 5-FU with or without LV and dipyridamole has been explored in clinical trials. With 175 mg/m2 dipyridamole orally every 6 hours, mean peak and trough free drug concentrations are 38 and 23 nmol/L, respectively, which are much lower than the optimal concentrations in cell culture models.482With CI of 285 mg/m2 per day of dipyridamole for 3 days (the highest tolerated dose), the mean Css of total and free dipyridamole is 6.7 µmol/L and 24 nmol/L, respectively.483 In paired patient courses of CI 5-FU, with or without dipyridamole, dipyridamole administration was associated with significantly lower 5-FU Css and a faster clearance.210, 216 Thus, the relatively high concentrations of free dipyridamole needed to optimally modulate 5-FU toxicity, metabolism, and DNA damage in tissue culture systems are not clinically achievable with systemic administration. The achievable Css of free dipyridamole with infusional or high-dose oral therapy may be sufficient to modulate the transport of dThd and other nucleosides in healthy tissues and some tumor tissues.483, 484 However, orally administered dipyridamole failed to improve the activity of a 5-FU and LV regimen in a randomized trial in patients with advanced colorectal cancer.485

Interaction of 5-Fluorouracil with Platinum Analogs

Synergism between cisplatin and 5-FU has been demonstrated in preclinical models in vitro and in vivo,486, 487, 488, 489, 490, 491, 492 and preclinical studies point to enhancement of DNA-directed toxicity as the mechanism. In some models, the toxicity of 5-FU and cisplatin is abrogated by dThd but potentiated by LV.486, 489, 490 In an ovarian cancer cell line, a 1-hour incubation with cisplatin (10 µmol/L) increased FH and CH FH4 pools by 2.5-fold and increased ternary complex formation by the same magnitude.486 The apparent basis is cisplatin-mediated inhibition of methionine uptake, which stimulates the endogenous synthesis of methionine from homocysteine and increases the conversion of 5-CH FH to FH, which is a precursor of 5,10-CH FH (Fig. 7-8). Cisplatin-mediated inhibition of intracellular L-methionine metabolism accompanied by expansion of the reduced folate pool has been confirmed in vivo.492 Other effects of cisplatin on DNA integrity or interactions with cell surface nucleic acids and plasma membrane also may be important. Enhanced DNA damage and inhibition of the repair of cisplatin-induced DNA interstrand cross-links have been noted with the combination.488, 490

In some models, concurrent exposure to both drugs is efficacious,486, 589 whereas other models report that preexposure to 5-FU before cisplatin administration is superior to the opposite sequence.487, 488, 490 In a human squamous cancer cell line, optimal cytotoxicity was seen with a 24-hour preexposure to 5-FU, followed by cisplatin after a 24 to 48-hour drug-free interval; the removal of cisplatin-induced DNA interstrand cross-links was significantly reduced compared with cells exposed to cisplatin alone or to 5-FU followed immediately by cisplatin.488 The lag time for 5-FU effects and the inability of dThd to reverse the interaction raised the possibility that RNA-directed effects might be involved. 5-FU inhibits ERCC1 and γ-glutamylcysteine synthetase mRNA expression in a cisplatin-resistant human squamous carcinoma cell line, suggesting that 5-FU-mediated interference with the expression of DNA repair enzymes might enhance DNA damage associated with cisplatin exposure.72 Preexposure of NCI H548 colon cancer cells to 5-FU for 24 hours followed by cisplatin for 2 hours produced more than additive cytotoxicity and a greater degree of single-stranded-DNA fragmentation in parental and nascent DNA compared with the opposite sequence.488

Although phase 2 studies suggested a beneficial effect of 5-FU plus cisplatin in colorectal carcinoma, randomized studies comparing bolus or CI 5-FU with or without bolus cisplatin indicated that the clinical toxicity was increased without improvement in overall disease control.493, 494, 495 Cisplatin is inactive as a single agent in colorectal cancer, and the necessary cellular events allowing a positive interaction are not present in this tumor type. In contrast, the combination of 5-FU and cisplatin has shown promising results in diseases in which both agents have single-agent activity, including squamous cell cancers arising in the anus, head and neck, esophagus, and cervix. The influence of sequence and timing of cisplatin and 5-FU administration in determining the extent of therapeutic effect, toxicity, or both, has not been carefully studied in clinical trials.

Oxaliplatin has also shown additive or synergistic cytotoxic properties with 5-FU in vitro and in vivo.496, 497, 498, 499 Decreased catabolism of 5-FU and down-regulation of thymidylate synthase expression are possible explanations for the synergy.498, 499 Unlike cisplatin, oxaliplatin has single-agent activity in colorectal cancer. Responses have been seen when oxaliplatin is added to a 5-FU-based regimen on which patients have had documented disease progression, suggesting clinical synergy. Randomized trials suggest a substantial improvement in the response rate when oxaliplatin is added to 5-FU and LV in advanced colorectal cancer and 3-year, disease-free survival as adjuvant therapy for colon cancer.500, 501

5-Fluorouracil and Taxanes

Paclitaxel is a taxane derivative that binds to the β-subunit of tubulin in the microtubule and promotes the formation of extremely stable microtubules. Antagonism between paclitaxel and 5-FU has been described in vitro.502, 503, 504 Sequential 24-hour exposures to paclitaxel followed by 5-FU were additive in four human cancer cell lines using the MTT assay, whereas the opposite sequence was subadditive in three of the four cell lines.502 Concurrent exposure of BCap37 breast cancer cells and KB cells to 100 nmol/L paclitaxel and 10 µmol/L 5-FU inhibited the customary oligonucleosomal-DNA fragmentation seen with paclitaxel alone at 48 and 72 hours.503 In this model, 5-FU diminishes the ability of paclitaxel to produce G2-M blockade and prevents apoptosis.

In MCF-7 breast cancer cells, 24-hour exposures to 5-FU and paclitaxel in various sequences suggested that preexposure to 5-FU, followed by paclitaxel, resulted in marked antagonism, whereas sequential paclitaxel followed by 5-FU was optimal.504 Concurrent or preexposure to paclitaxel did not affect [3H]5-FU metabolism, [3H]5-FU-RNA incorporation, or the extent of 5-FU-mediated TS inhibition. Paclitaxel led to G2-M phase accumulation persisting for 24 hours after drug exposure, whereas a 24-hour 5-FU exposure produced S-phase accumulation. 5-FU preexposure diminished paclitaxel-associated G2-M phase block, whereas subsequent exposure to 5-FU after paclitaxel did not. 5-FU exposure resulted in transient induction of p53 and p21, which returned to basal levels 24 hours after drug removal. p53 and p21 protein content also markedly increased during paclitaxel exposure, accompanied by phosphorylation of Bcl-2. Pronounced DNA fragmentation was seen at 48 hours when cells were exposed to paclitaxel for an initial 24-hour period. Paclitaxel-associated DNA fragmentation was not prevented by concurrent or subsequent exposure to 5-FU. In this model, paclitaxel-mediated G2-M phase arrest appeared to be a crucial step in induction of DNA fragmentation. The potential importance of sequence of taxane and 5-FU administration has not been explored in clinical trials.

Camptothecins and 5- Fluorouracil

In preclinical models, CPT-11 given 6 to 24 hours prior to 5-FU or other TS inhibitors is the most effective sequence compared with concurrent or reverse sequences.505, 506, 507, 508, 509, 510 Because active DNA synthesis is required to convert the formation of covalent SN- 38-topoisomerase I-cleavable complexes to a cytotoxic lesion, inhibition of TS during or prior to formation of the SN-38 cleavable complex is antagonistic.

Interaction of 5-Fluorouracil with Ionizing Radiation

Heidelberger et al.511 discovered that growth-inhibitory doses of radiotherapy in rodent tumors were made curative by the addition of 5-FU, and ineffective regimens of 5-FU became active by the addition of a single dose of radiotherapy. The synergistic interaction has been confirmed by other investigators.512, 513,514, 515, 516, 517, 518, 519, 520, 521 Combined treatment with 5-FU and radiotherapy leads to concentration- and time-dependent enhancement of cell killing in HeLA and HT-29 cells.513, 514 Enhanced radiosensitization depends on 5-FU exposure for a period longer than the cell-doubling time. The optimal effects are observed when 5-FU continues for at least 48 hours after irradiation, with little or no synergy if 5-FU is given either before or for only 3 hours after irradiation. However, the optimal schedule for 5-FU radiosensitization in preclinical models varies depending on the model system used. In DU-145 prostate cancer cells, 5-FU modulation of radiosensitivity is apparent with either a 1-hour pulse of 100 µmol/L 5-FU plus irradiation at 30 minutes, or with continuous exposure to 4 µmol/L 5-FU and irradiation given either immediately before or 17 hours after 5-FU.515

In p53 mutant HT-29 cells, time-dependent radiosensitization occurs after a 2-hour exposure to 0.5 µmol/L FdUrd.518 TS is maximally inhibited at the end of FdUrd exposure, but TS inhibition persists for up to 32 hours after drug removal, accompanied by dTTP pool depletion. An increase in radiosensitivity, however, is not apparent until 16 hours after drug removal. The increase in radiation sensitivity parallels the gradual accumulation of cells in early S phase, a radiosensitive phase of the cell cycle. In another model, 8- or 24-hour preexposures to low concentrations of FdUrd enhance DNA damage by inhibiting repair of double-stranded breaks; the addition of LV and dipyridamole enhances FdUrd-mediated radiosensitization and the interference with DNA repair.516, 517Although HT-29 and SW-620 colon cancer cells have the same p53 mutation, the response to FdUrd-mediated radiosensitization is different.520 Exposure to 100 nmol/L FdUrd for 14 hours produces comparable inhibition of TS activity by 75 to 80%, yet the radiosensitive HT-29 cell line progresses into S phase, whereas the insensitive cell line arrests at the G1-S boundary. Although cyclin D protein levels do not change with FdUrd treatment in either cell line, cyclin E protein content increases by sevenfold to ninefold in both lines. Cyclin E-dependent kinase activity increases only in HT-29 cells, which may account for this cell line's progression into S phase.520 These findings suggest that a G1-S checkpoint that influences radiosensitization produced by FdUrd is not dependent on normal p53 function.

In vivo, the combination of 5-FU, with or without LV, with radiation on several schedules has proven effective in increasing the delay in tumor regrowth.511,512, 513, 514 The experimental evidence predominantly supports more prolonged exposure to fluoropyrimidines as optimal. The underlying mechanism(s) for this synergistic interaction may be influenced by schedule and duration of exposure. FdUMP-mediated inhibition of TS with resulting dTTP pool depletion, deoxyribonucleotide imbalance, increased DNA damage, inhibition of DNA repair, and accumulation of cells in S phase appear to be important features of radiosensitization. The RNA-directed effects of 5-FU might conceivably play a role, but have not been clearly implicated.

5-FU given alone or in combination with other agents during radiotherapy has demonstrated efficacy in patients with either squamous cell cancers arising in the anal canal, cervix, head and neck, and esophagus, or adenocarcinomas arising in the rectum.522, 523, 524, 525 Diverse schedules of 5-FU have been used, including bolus administration of 5-FU during the first and final 3 days of radiation, 96- to 120-hour CI for the first and last week of radiation, and CI throughout the entire radiation treatment. A randomized trial in patients with high-risk rectal cancer comparing 5-FU given by intermittent bolus injections with protracted CI during postoperative radiation therapy to the pelvis demonstrated significant improvements in time to relapse and survival in favor of the infusional 5-FU arm.525

ORALLY BIOAVAILABLE 5′-FLUOROPYRIMIDINES

The structures of selected oral 5-FU-prodrugs are shown in Figure 7-11. Two of the drugs, ftorafur and doxifluridine, were initially tested with intravenous administration, whereas the other drugs were developed as a strategy to permit oral administration. Features of the oral 5-fluoropyrimidine drugs are shown in Table 7-13.

Ftorafur and UFT

Ftorafur [1-(2-tetrahydrofuranyl)-5-fluorouracil, tegafur; MW= 200] is a furan nucleoside that has clinical activity against adenocarcinomas and is less myelosuppressive, but more neurotoxic, than 5-FU. Ftorafur is a prodrug and is slowly metabolized to 5-FU by two major metabolic pathways.526, 527, 528, 529,530, 531 One pathway is mediated by microsomal cytochrome P-450 oxidation at the 5′-carbon of the tetrahydrofuran moiety, resulting in the formation of a labile intermediate (5′-hydroxyftorafur) that spontaneously cleaves to produce succinaldehyde and 5-FU (Fig. 7-12).528 Studies with human liver microsomes indicate that cytochromes P450 1A2, 2A6, and 2C8 contribute to the biotransformation of tegafur into 5-FU.530 The second pathway occurs in the cytosol, and is thought to be mediated by thymidine phosphorylase.529, 531 Enzymatic cleavage of the N-1-C-2′ bond to yield 5-FU and 4-hydroxybutanal; the latter undergoes further enzymatic conversion to form γ-butyrolactone (γ-BL), BL, or γ-hydroxybutyric acid (γ-HB); succinaldehyde is partially converted to these latter two compounds. In vivo, the liver is the major source of cytochrome P-450, with lower levels in the GI tract and much lower levels in the brain. In vitro studies with tissue homogenates from liver, GI tract, and the brain containing the soluble enzyme pathway have documented metabolism of ftorafur to 5-FU. Small amounts of 3′- and 4′-hydroxyl derivatives have been isolated from urine.526, 527 After intravenous bolus injection of 1 g/m2, ftorafur and a major metabolite, dehydroftorafur, were detected in serum, whereas 5-FU was not.532 The uniformly low 5-FU Cp in pharmacokinetic studies suggest that metabolic conversion of ftorafur to 5-FU occurs intracellularly, without subsequent redistribution via the systemic circulation.533, 534, 535 Thus, 5-FU Cp may not accurately reflect the extent of this intracellular conversion.

Figure 7.11 Structures of orally administered 5-fluoropyrimidine analogs. BOF-A2, emitefur, 3-{3-[6-benzoyloxy-3-cyano-2-pyridyloxycarbonyl]benzoyl}-1-ethoxymethyl-5-fluorouracil.

 

The pharmacokinetic behavior of ftorafur has been described for intravenous and oral routes of administration. After intravenous bolus injection, ftorafur undergoes an initial distribution phase, followed by a prolonged t1/2 ranging from 6 to 16 hours.526, 527, 533, 534, 536 The clearance is approximately 31 mL/min per square meter, and the Vd (15 to 30 L/m2) approximates that of total-body water. After oral administration, absorption is virtually 100%. After 2 g/m2orally, ftorafur appears in plasma by 11 minutes, and Cmax occurs at 3.2 hours.535 Simultaneous sampling of blood from portal and peripheral veins indicates that ftorafur appears sooner in the portal vein; peak levels in the peripheral vein occur 1.7 hours later, consistent with rapid absorption and hepatic retention.

Ftorafur has been administered intravenously in doses of 1.50 to 2.25 g/m2 daily for 4 or 5 days or single doses of 4 g/m2 weekly.537, 538, 539, 540, 541, 542 The primary clinical toxicities with these schedules are GI symptoms (diarrhea, cramps, vomiting, and mucositis) and neurologic side effects (altered mental status, cerebellar ataxia, and, rarely, coma). The neurotoxicity has been attributed to the high concentrations of parent drug found in the CSF.526, 533, 534 The ftorafur metabolite γ-hydroxybutyrate occurs physiologically in brain and CSF, has anesthetic properties, produces concentration-dependent CNS depression, and may contribute to neurotoxicity. A recommended oral dose is 1.5 g/m2 daily for 14 to 21 days, although some investigators suggest that a less-intensive regimen of 0.8 to 1.0 g/m2 daily (in divided doses) for 14 of 28 days is better tolerated.539, 540 GI side effects are predominant with the oral route, while neurotoxicity (dizziness and lethargy) occurs infrequently.

Phase 2 trials in a variety of solid tumors suggest that ftorafur has activity consistent with that expected with 5-FU.537, 538, 539, 540, 541 Some patients failing 5-FU-containing regimens have responded to a protracted oral schedule of ftorafur.540, 542 A randomized trial indicated similar antitumor activity for single-agent ftorafur and 5-FU, whereas toxicity profiles differed.542

The option for oral administration has maintained interest in the use of ftorafur. Oral ftorafur has been combined with oral LV on a 21-day schedule; the recommended dose is 1,600 mg in three divided doses with 500 mg LV in five divided doses.543 UFT, a combination of uracil and ftorafur (molar ratio of 4:1), entered into clinical trials in Japan in the early 1980s. Preclinical studies indicate that UFT results in significantly higher tumor-to-serum 5-FU ratios than observed with ftorafur alone.532, 544 UFT is usually given orally in divided doses daily for either 5 or 28 days. With oral doses ranging from 50 to 300 mg/m2, maximum Cp of ftorafur and 5-FU occur between 0.6 and 2.1 hours; ftorafur levels (13.5 to 100 µmol/L) greatly exceed 5-FU levels (0.2 to 7.0 µmol/L), and ftorafur clearance is approximately 70 mL/min per square meter.545 Intratumoral 5-FU levels were 2.3-fold higher than in healthy kidney tissue in patients undergoing nephrectomy for renal cell carcinoma 1 day after a 5-day course of twice-daily ftorafur.546 Another study reported that the maximum 5-FU concentration in bladder cancer tissue was fourfold and 10-fold higher than in healthy bladder epithelium and peripheral blood, respectively.547 An interesting preclinical study reported that (-HB and 5-FU, both metabolites of ftorafur and UFT, inhibit the angiogenesis induced by vascular endothelial growth factor.548

TABLE 7.13 ORALLY ADMINISTERED 5-FLUOROPYRIMIDINES

Agents

Pharmacologic Effect

Common Clinical Schedules

UFT, 2-drug combination: uracil and ftorafur (4:1 molar ratio)
Orzel = UFT + calcium leucovorin

Prodrug containing Uracil, a competitive inhibitor of DPD; Ftorafur is an oral fluorouracil prodrug.

UFT 300 mg/m (2 + LV 75–150 mg p.o. daily in 3 divided doses for 28 of 35 d
or
LV 500 mg/m2 IV + UFT 195 mg/m (2 p.o. d 1 then oral LV 15 mg + UFT 195 mg/m2 q 12 hr for 14 of 28 d

Eniluracil with oral 5-FU

Eniluracil is a mechanism-based inhibitor of DPD;
It renders 5-FU bioavailability near 100%;
It prevents formation of 5-FU catabolites.

Eniluracil 20 mg p.o. + 1 mg/m (2 5-FU p.o. twice daily for 28 of 35 d

S-1, 3-drug combination:
   Ftorafur 5-chloro-2,4-dihydroxypyridine


Ftorafur is an oral fluorouracil prodrug. 5-chloro-2,4-dihydroxypyridine is a potent, competitive inhibitor of DPD.

40 mg/m2 (40–60 mg) p.o. twice daily for 28 of 42 d
or
30 mg/m2 p.o. twice daily for 28 of 35 d

Potassium oxonate 1:0.4:1 molar ratio

Potassium oxonate is a competitive inhibitor of orotate phosphoribosyl transferase (decreases 5-FU anabolism and gastrointestinal toxicity).

 

Capecitabine (xeloda)

Oral 5-FU prodrug.
Parent drug absorbed intact.
Converted sequentially to 5′-deoxy-5-fluorocytidine, 5′-deoxy-5-fluorouridine, and 5-FU.
5-FU liberated by thymidine phosphorylase.

2,500 mg/m2 p.o. daily in 2 divided doses for 14 of 21 d

BOF-A2 (emitefur)
   1-ethoxymethyl-5-fluorouracil 3-cyano-2,6-dihydroxypyridine


Masked oral 5-FU prodrug.
Parental drug absorbed intact.
5-FU liberated by hepatic microsomal enzymes.
3-cyano2, 6dihydroxypyridine is a potent competitive inhibitor of DPD.

200 mg p.o. twice daily for 14 of 28 d
or
200 mg/m2 p.o. + LV 60 mg p.o. in 2 divided doses daily for 14 of 21 d

BOF-A2, emitefur, 3{3[6benzoyloxy3cyano2pyridyloxycarbonyl]benzoyl}-1-ethoxymethyl-5-fluorouracil; DPD, dihydropyrimidine dehydrogenase; 5-FU, 5-fluorouracil; LV, leucovorin; UFT, uracil and ftorafur.

Combined phase 2 data from 438 patients revealed that UFT had activity in cancers arising in the stomach (28%), pancreas (25%), gallbladder and bile duct (25%), liver (19%), colon and rectum (25%), breast (32%), and lung (7%).549 Hematologic toxicity was mild; GI toxicity included anorexia (24%), nausea and vomiting (12.5%), and diarrhea (12%).

Comparison of 5-FU pharmacokinetics with equimolar total daily doses of UFT and CI 5-FU indicated that during the first day, the Css and AUC(0-8h) were 1.8- and 1.7-fold higher with CI 5-FU.551 By day 5, however, these parameters were comparable. With a 28-day schedule followed by a 2-week break, administration of UFT in three divided doses every 8 hours was much better tolerated than single-daily or twice-daily dosing.552 Ftorafur clearance is saturable, resulting in disproportionate increases in the AUC and toxicities with increasing dose levels. A daily dose of 400 mg/m2 given in three divided doses was recommended on this schedule. A phase 2 study of UFT (300 to 350 mg/m2) orally plus LV (150 mg orally) in three divided doses daily for 28 days revealed a 42% response rate in 45 patients with previously untreated colorectal cancer.552 With this schedule, GI toxicity (anorexia, nausea, vomiting, and diarrhea) is generally mild to moderate in severity. Hematologic toxicity is mild, and symptomatic hand-foot syndrome is uncommon. A different schedule used a single intravenous dose of LV (500 mg/m2) followed by oral UFT (195 mg/m2) on day 1, followed by oral LV (15 mg) and UFT (195 mg/m2) every 12 hours on days 2 through 14, followed by a 2-week rest.553 The response rate as first-line therapy in 75 patients with advanced colorectal cancer was 39%. The primary toxicity was GI, but was of grade 3 to 4 severity in only 3.5%; hematologic toxicity was minimal, and the regimen was safe in older patients.

Figure 7.12 Metabolism of ftorafur. 5-FU, 5-fluorouracil.

The extent of TS inhibition was determined in tumor tissue taken from patients with gastric cancer assigned treatment with UFT alone (400 mg ftorafur per day) or with LV (30 mg/day) in divided doses every 12 hours for 3 days before gastrectomy, with the last dose 6 hours before surgery.554 TS inhibition was significantly greater in eight patients treated with UFT and LV compared with that measured in nine patients receiving UFT alone (61% versus 32% inhibition).554

A bioavailability study compared the pharmacokinetics of UFT and LV in 18 patients after UFT alone, LV alone, or a combination of the two.555 When LV was coadministered with UFT, there were no significant effects on tegafur, uracil, or 5-FU Cmax or AUC; no significant differences were seen in LV and 5-methyltetrahydrofolate plasma levels after LV alone or with UFT. As might be expected, interpatient variability in UFT and LV pharmacology was pronounced.

No randomized trials have directly evaluated the benefit of adding LV to UFT. In Western countries, a proprietary combination of UFT and oral calcium LV (Orzel) showed activity in phase 2 studies.556 A monthly schedule was selected for randomized trials owing to the higher projected dose intensity (2,100 versus 1,365 mg/m2 per week) and excellent safety profile. The results of two large phase 3 trials comparing UFT plus LV with the monthly schedule of bolus 5-FU plus LV in patients with metastatic colorectal cancer suggest comparable efficacy (response rates, 12% versus 15% and 11% versus 9%), but a more favorable safety profile with significantly fewer episodes of febrile neutropenia and infection.557, 558 Because the sponsor could not provide data supporting the contribution of each of the individual components of Orzel, it was not approved in the United States.

 

5′-Deoxy-5-Fluorouridine

The synthetic fluoropyrimidine 5′-deoxy-5-fluorouridine (5′-dFUrd, doxifluridine, Furtulon; MW= 246) has shown increased specificity for tumor cells as compared with healthy tissues in some preclinical models.559, 560, 561, 562, 563, 564 Because the 5′-carbon of the ribose moiety lacks a hydroxyl group, 5′-dFUrd cannot serve as a substrate for Urd kinase. Urd and dThd phosphorylase are potentially capable of liberating 5-FU by cleaving the glycosidic bond. 5-FU is thus released intracellularly and can undergo further metabolic activation.559, 560, 561, 562, 563, 564, 565 Urd phosphorylase primarily cleaves pyrimidine ribonucleosides but also cleaves pyrimidine 2′- and 5′-deoxyribonucleosides. In contrast, dThd phosphorylase is thought to be relatively specific for pyrimidine 2′- and 5′-deoxyribonucleosides.15, 16, 564, 565, 566, 567, 568 Urd phosphorylase is present in virtually all healthy and tumor tissues studied, whereas the activity of dThd phosphorylase is much more variable in human and rodent tumors.15, 16, 469, 562, 567, 568 Distinct differences exist between the enzymes isolated from human and mouse tissues in terms of biologic properties, substrate specificities, and their roles in the metabolism of endogenous pyrimidine nucleosides and their 5-fluorinated analogs.565 Substrate specificity also varies between enzymes from different human tissues, suggesting the presence of isoenzymes. In human liver, dThd phosphorylase contributes from 99 to 100% of the phosphorolysis of 5′-dFUrd and FdUrd, whereas the contribution from dThd phosphorylase isolated from mouse liver (73% and 83%, respectively) or human placenta (86% and 93%) is lower.565

5′-dFUrd shows selective cytotoxicity against tumor tissues and relatively low toxicity against healthy tissues, presumably because of greater enzymatic activation in neoplastic tissues than in healthy tissues.561, 563 When 5′-dFUrd is used as the substrate, human and rodent tumor tissue (including esophagus, stomach, intestine, pancreas, breast, urinary bladder, and lung) usually contains higher specific activity of pyrimidine phosphorylase(s) than do healthy tissues from the same organs, suggesting potentially selective cytotoxicity.562, 564 In contrast, nonmalignant human liver tissue has much higher activity than healthy tissues of other digestive organs, but its activity is either comparable with or higher than that in malignant tissues from various origins.564

The cytotoxicity of 5′-dFUrd in vitro correlates with activity of the pyrimidine phosphorylases.559, 560, 561 Comparison of the toxicity of 5′-dFUrd in human tumor cells and human bone marrow in vitro indicates that 5′-dFUrd, but not 5-FU or FdUrd, has selective tumor toxicity.561 Several investigators have reported that the antitumor activity of 5′-dFUrd against human tumor xenografts does not correlate with the ability of tumor homogenates to convert [3H]5′-dFUrd to 5-FU, suggesting that the liver may be the major site of metabolic activation of this prodrug in vivo.563, 564

Pharmacokinetic studies after intravenous administration by either 30- or 60-minute infusions reveals nonlinear elimination: 5′-dFUrd metabolism is saturable at plasma levels above 40 to 50 µmol/L; a fall in clearance of the drug occurs with increasing dose.569, 570 With low doses (1 to 2 g/m2 over 30 minutes), the disappearance of 5′-dFUrd follows first-order kinetics. With higher dosages (15 g/m2), the Cmax is about 175 µmol/L, with a primary t1/2 of 25 minutes.570With rapid intravenous injection of 2 or 4 g/m2, the clearance falls from 330 to 200 mL/min per square meter. The peak plasma levels of 5-FU are much lower than 5′-dFUrd levels, and the ratio is influenced by the infusion rate.571, 572, 573, 574 Urinary excretion is virtually 100%; unchanged drug and FBAL account for the majority of the compounds.571, 572 After intravenous bolus, the renal clearance of 5′-dFUrd (166 mL/min per square meter) exceeds the expected glomerular filtration rate, suggesting that 5′-dFUrd undergoes renal tubular secretion; however, there is no evidence that renal clearance is saturable.570 The cumulative biliary excretion has been estimated to be 0.8% of the injected dosage; a FBAL-bile acid conjugate is the major biliary metabolite, and FBAL accounts for 10%.572 When 5′-dFUrd is administered by CI over 5 days (0.75 to 4 g/m2 per day), nonrenal clearance is not saturable; Css levels range from 0.7 to 26.5 µmol/L, and increase linearly with dose.571 Nonrenal clearance averages 728 mL/min per square meter and is about seven times higher than renal clearance.

Initial phase 1 testing indicated that myelosuppression and stomatitis were dose-limiting at 4,000 mg/m2 per day for 5 days intravenously; CNS toxicity and ECG changes were also noted.574 With a 6-hour intravenous infusion weekly-for-3-weeks schedule of 5′-dFUrd, neurotoxicity is dose-limiting with 10 to 12.5 g/m2 per week.575 Nausea and vomiting, diarrhea, and cutaneous reactions occur with both schedules. A 5-day CI of 5′-dFUrd is well tolerated at daily doses of 3.5 g/m2 or less, except for mild nausea; dose-limiting toxicities at 4 g/m2 per day include neutropenia, thrombocytopenia, mucositis, and rash.573

Phase 2 studies using the weekly 6-hour infusion schedule demonstrated responses in breast cancer (36%) and colorectal cancer (22%).575 eurologic toxicity (dizziness, ataxia, and alterations of consciousness) was prominent, occurring in 42% of the patients, with four lethal events. 5′-dFUrd is active in colorectal, breast, ovarian, and head and neck cancer when given by rapid intravenous infusion on a daily-for-5-days schedule, but was accompanied by a high incidence of dose-related neurotoxicity reminiscent of Wernicke-Korsakoff syndrome.577, 578, 579 A small randomized trial comparing a 5-day course of either 5-FU (450 mg/m2 per day) with 5′-dFUrd (4,000 mg/m2 per day) was interrupted because of cardiac toxicity (chest pain, arrhythmias, and ventricular fibrillation) and neurotoxicity (48%) on the 5′-dFUrd arm.577 The response rate favored the 5′-dFUrd arm (20% of 25 patients versus 7% of 27 patients with 5-FU). Cardiac toxicity was observed sporadically in other clinical trials.

Lengthening the infusion of 5′-dFUrd to 1 hour reduces the incidence and severity of neurotoxicity to 16 to 23%. Two randomized trials compared a 1-hour infusion of either 5′-dFUrd (4 g/m2 per day) or 5-FU (450 or 500 mg/m2 per day) daily for 5 days in advanced colorectal cancer.580, 581 The response rate was higher with 5′-dFUrd on both trials (23% of 31 and 5% of 112 patients, respectively) compared with 5-FU (7% of 30 and 1% of 110 patients, respectively), and the time to disease progression favored the 5′-dFUrd arm in one study (48 versus 39 weeks, P= .02); survival was not improved on either trial. The arms were not equitoxic; the 1-hour infusion of 5-FU was relatively nontoxic, and dose escalation was not allowed.

Unlike 5-FU, 5′-dFUrd is well absorbed by the oral route. An oral regimen of 5′-dFUrd 1,200 mg daily for 28 days is well tolerated, with mild-to-moderate diarrhea, nausea, and vomiting as the principal side effects; higher doses were complicated by a higher incidence of diarrhea.582 Another commonly used oral regimen is l-LV 25 mg followed 2 hours later by 5′-dFUrd 1,200 mg/m2 daily for 5 days followed by 5 days of rest. With this schedule, Cmax values for 5′-FdUrd and 5-FU average approximately 67 and 6 µmol/L, respectively.583 Among 62 previously untreated patients with colorectal cancer, 32% responded, as did 13% of patients with prior systemic-5-FU therapy.583

Capecitabine

Capecitabine [N (4-pentoxycarbonyl-5′-deoxy-5-fluorocytidine, Xeloda] is the first oral 5-FU prodrug to be approved in the United States, on the basis of its activity in patients with metastatic breast cancer whose disease is refractory to two earlier regimens.584 This agent is absorbed intact as the parent drug through the GI mucosa. It then undergoes a three-step enzymatic conversion to 5-FU (Fig. 7-13). In the liver, 5′-deoxy-5-fluorocytidine (5′-dFCyd) formation is catalyzed by carboxylesterase (CES), which is mainly expressed in microsomes, but a cytosolic carboxylesterase, CES1A1, also contributes to formation of 5′-dFCyd.585, 586 Cytidine deaminase, a widely distributed enzyme, produces 5′-dFUrd, and dThd phosphorylase then generates 5-FU. Clinical studies have documented rapid GI absorption of the parent drug with efficient conversion to 5′-dFUrd; 5-FU Cp are low.587, 588, 589

Figure 7.13 Activation of capecitabine. The enzymes are 1, carboxylesterase; 2, cytidine deaminase; 3, thymidine phosphorylase. 5-FU, 5-fluorouracil.

Several preclinical studies have documented preferential accumulation of 5-FU in tumor tissue compared with healthy tissue.590, 591, 592, 593, 594 Intracellular accumulation of 5-FU was studied in four human-cancer xenografts after administration of either oral capecitabine (1.5 mmol/kg) or intraperitoneal 5-FU (0.15 mmol/kg) at their maximum tolerated doses (MTD) on a daily for a 7-day schedule.591 With capecitabine, the median AUC of 5-FU in tumor tissue was 250 nmol/hr per gram, 120-fold higher than the plasma AUC. After 5-FU, the median 5-FU AUC in tumor tissue was 12.2 nmol/hr per gram, a twofold increase over the plasma AUC. Despite the 10-fold higher dose of capecitabine, the 5-FU AUC in plasma was one-third of that observed with intraperitoneal 5-FU. This study provides strong evidence that 5-FU is preferentially formed in tumor tissue versus plasma after capecitabine administration.

Eighteen (75%) of 24 human cancer xenografts were sensitive to capecitabine.592 Among 15 tumors with dThd-phosphorylase activity greater than 50 µg/mg per hour, 87% were sensitive, but 56% of tumors with lower enzyme activity were also sensitive.592 However, tumors with relatively low ratios of dThd phosphorylase to DPD activity were more likely to be resistant, whereas tumors with higher ratios were uniformly sensitive. Thus, tumors with low dThd-phosphorylase activity may still retain sensitivity to capecitabine provided the activity of DPD is also low, whereas capecitabine might not be effective in tumors with higher dThd phosphorylase if the DPD activity is also high. Measurement of dThd phosphorylase and DPD by ELISA assay in 241 human tumor specimens indicated that the ratio of dThdPase:DPD was high (median ratio of >1.5) in esophageal, renal, breast, colorectal, and gastric cancers.595

The activities of carboxylesterase, cytidine deaminase, and dThd phosphorylase were measured in human tumor and adjacent healthy tissue surgically resected from patients with a variety of cancers.590 Using capecitabine as the substrate, carboxylesterase activity was almost exclusively localized in human-liver and hepatocellular carcinoma, with minimal activity in other tumors and organs, including the intestinal tract and plasma. Homogenates prepared from most healthy and tumor tissues were able to deaminate 5′-dFCyd, although healthy liver had the highest activity. With 5′-dFUrd as substrate, dThd-phosphorylase activity was detected in all healthy tissues, with the highest activity in liver tissue. dThd-phosphorylase activities showed much greater variability in tumor tissue; with few exceptions, the activity was higher in tumor tissue obtained from 11 different sites of origin than that of the corresponding healthy tissues.

Two schedules have been evaluated in the clinic: a continuous schedule for 28 days (MTD 1,600 mg/m2 orally daily), and a daily for 14 days every 3-weeks schedule (MTD 3,200 mg/m2 orally daily).587, 590 Capecitabine is given as two equal doses approximately12 hours apart, taken within 30 minutes after a meal. The toxicity profile favors the daily for 14 of 21-day schedule, with a recommended total daily dose of 2,500 mg/m2 orally. When given with low-dose oral LV (60 mg orally daily), the recommended total daily dose is 1,650 mg/m2 for 14 of 21 days.596 Dose-limiting toxicities include diarrhea, nausea, vomiting, and hand-foot syndrome; myelosuppression is uncommon. With more widespread use, capecitabine-associated cardiac, ocular, and neurologic toxicity has been reported.597, 598, 599

Preferential accumulation of 5-FU in primary colorectal tumors compared with adjacent healthy tissue has been documented after oral administration of capecitabine to patients. The ratio of 5-FU concentration in tumor tissue was about 3.2-fold higher than healthy tissue, and the mean tissue:plasma 5-FU concentration ratios exceeded 20 for colorectal tumor.600 These results could be explained by the fourfold higher activity of dThd phosphorylase in colorectal tumor tissue. Another study reported the importance of the ratio of dThd phosphorylase and DPD levels in primary colorectal cancer as correlates of benefit to adjuvant 5′-dFUrd therapy.601

The pharmacokinetics of capecitabine and its metabolites have been measured using liquid chromatography mass spectrometry in studies by the pharmaceutical sponsor.588, 602, 603, 604, 605 After an initial dose of 1,255 mg/m2 (total daily dose 2,510 mg/m2), peak Cp of parent drug, the two nucleoside metabolites, 5-FU and FBAL are reached about 2 hours after dosing. The t1/2 is about 1 hour for all metabolites except for FBAL, which has an initial t1/2 of 3 hours. The AUC of 5′-dFUrd is the greatest and exceeds the AUC (units= microgram % hour per milliliter) of 5-FU by 12-fold. Over the dosage range used clinically, there is no evidence of dose dependency in the pharmacokinetic parameters. No appreciable accumulation of either parent drug or metabolites is noted when comparing pharmacokinetic values from days 1 and 14, other than a 22% higher 5-FU AUC on day 14, suggesting a change in 5-FU clearance with time. The low Cp of 5-FU supports the notion that its formation primarily occurs within cells.

The clinical safety of capecitabine has been determined exclusively with capecitabine taken within 30 minutes after a meal. Comparison of capecitabine pharmacokinetics before and after food intake indicates a profound effect on the Cmax of capecitabine and most of its metabolites. The AUC of capecitabine is 1.5-fold higher when taken before food; a moderate effect is also noted for 5′-dFCyd, with a 1.26-fold higher AUC before food, and food ingestion has only a minor influence on the AUC of the other metabolites.602, 603 This reinforces the recommendation to ingest capecitabine within 30 minutes following food. In patients with hepatic dysfunction, Cp of capecitabine, 5′-dFUrd, 5-FU, DHFU, and FBAL were higher than in those with normal function, while the opposite was found for 5′-dFCyd.604 These effects did not appear to be clinically significant, and it is recommended that although caution should be used when treating patients with moderately impaired hepatic function, but there is no a priori need for dose reduction.604 In a small study, the AUC of 5′-dFUrd was higher in those patients with impaired renal function, and this increased correlated with an excess risk of severe toxicity.605 Based on these results, the sponsor recommends that patients with severe renal dysfunction should not be treated with capecitabine. In addition to the pharmacokinetic results, information from the clinical safety database led to the recommendation that patients with moderate renal impairment (30-50 mL/min based on a 24-hour urine collection) should be treated with 75% of the recommended standard starting dose to achieve systemic exposure comparable with that in patients with normal renal function.

Other analytic methods that have been developed to measure capecitabine and its metabolites in biologic samples, including liquid chromatography with mass selective detection for analysis of parent drug and nucleosides, gas chromatography/mass selective detection to measure 5-FU and FBAL, liquid chromatography with UV detection, fluorine-19 magnetic resonance spectroscopy, and capillary electrophoresis.606, 607, 608, 609

Two large randomized phase 3 trials comparing capecitabine with the monthly schedule of 5-FU and LV (425 mg/m2 and 20 mg/m2 daily for 5 days, respectively) in patients with advanced colorectal cancer have been conducted.610, 611 One trial involving 605 patients demonstrated a response rate in favor of capecitabine (23% versus 16%).610 Grade 3 to 4 toxicities included hand-foot syndrome (18%) and diarrhea (15%) for capecitabine and neutropenia (26%), mucositis (16%), and diarrhea (14%) for 5-FU and LV. A second international trial using an identical design involved 602 patients; the response rate favored capecitabine (27% versus 18%).611 Hand-foot syndrome and diarrhea of grade 3 to 4 severity occurred in 16% and 10% of capecitabine patients, whereas severe or worse neutropenia, mucositis, and diarrhea occurred in 20%, 13%, and 10% of patients receiving 5-FU and LV. Current strategies focus on combining capecitabine with agents that might induce thymidine phosphorylase in tumor tissue, such as ionizing radiation and cytokines.612, 613, 614, 615

Eniluracil Combined With 5-Fluorouracil

The uracil analog eniluracil (776C85, 5-ethynyluracil) is an extremely potent mechanism-based inactivator of DPD, and is ninefold more potent than bromvinyluracil.616 On binding of eniluracil to DPD (apparent K, 1.6 µmol/L), an unstable intermediate is formed, after which the drug becomes covalently linked to the enzyme through modification of an amino acid residue (Fig. 7-14).616 Administration of eniluracil to animals and humans results in complete inhibition of DPD throughout the body, as evidenced directly by enzyme assays and indirectly by up to 100-fold elevations of plasma-uracil levels.617, 618, 619When given with eniluracil, renal excretion of 5-FU becomes the predominant route of elimination. Oral administration of 5-FU with eniluracil renders 5-FU completely bioavailable.

Although eniluracil appears to be nontoxic when given alone, it shifts the 5-FU dose toxicity-response curves to lower doses. The combination of eniluracil (1 mg/kg per day) with 5-FU at one-tenth the single-agent dose produced complete tumor regressions in rats that are sustained for at least 90 days posttherapy.619 These results are superior to that seen with maximum 5-FU doses given either bolus daily for 5 days or with CI for 4 days, suggesting that the improved antitumor activity is not simply the result of prolonged 5-FU plasma exposure. To test the hypothesis that 5-FU catabolites may attenuate the antitumor activity of 5-FU, the antitumor activity of three regimens was compared in the rat model: 5-FU alone (100 mg/kg), eniluracil (1 mg/kg) followed by 5-FU (10 mg/kg), and eniluracil (10 mg/kg) followed by 5-FU (10 mg/kg) and DHFU (90 mg/kg). The regimen was repeated weekly for 3 weeks on all arms. The complete response rate was 13% with 5-FU alone, 94% with eniluracil plus 5-FU, and 38% with the three-drug combination, indicating that administration of DHFU interfered with the efficacy of eniluracil plus 5-FU.620

5-FU metabolism was monitored in an isolated rat liver perfusion model with fluorine-19 nuclear magnetic resonance spectroscopy during perfusion with 5-FU alone (15 mg/kg) or 5-FU preceded by eniluracil (0.5 mg/kg). Eniluracil produced a 27-fold decrease in the formation of catabolites and a sevenfold increase in anabolite formation.621 Eniluracil prevented the formation of the toxic catabolites FBAL, fluoroacetate, and 2-fluoro-3-hydroxypropionic acid, which have been implicated in 5-FU- associated neurotoxicity and cardiac toxicity. In mice bearing murine colon 38 tumors, ex vivo measurements of tissue extracts from liver, kidney, and tumor indicate a greater than 95% elimination of FUPA and FBAL signals in the tissues of mice that received 2 mg/kg of eniluracil before administration of 5-FU.622 A prolonged presence of 5-FU and increased formation of fluoronucleotides was noted in healthy and tumor tissues. Eniluracil prevented neurotoxicity associated with a CI of 5-FU in a canine model, suggesting that eniluracil-mediated inhibition of DPD prevents the formation of potentially toxic catabolites.309

Figure 7.14 Interaction between eniluracil and dihydropyrimidine dehydrogenase. E, dihydropyrimidine dehydrogenase; NADP, nicotinamide adenine dinucleotide; NADPH, nicotinamide adenine dinucleotide phosphate.

Clinical studies of oral eniluracil given once daily for 7 days at 0.74, 3.7, or 18.5 mg/m2 indicated that DPD activity in PBMCs was inactivated within 1 hour and remained inhibited by 93 to 98% 24 hours after dosing.623 Fourteen days after eniluracil, mean DPD activity was approximately 60% (0.74 mg/m2), 70% (3.7 mg/m2), and 125% (18.5 mg/m2) relative to baseline values. Because lower dosages of eniluracil would be expected to produce faster recovery, these observations suggest potential interpatient variability in the duration of DPD inhibition.

Pharmacokinetic comparison of 5-FU (10 mg/m2) given either intravenously or orally on day 2 with oral eniluracil (3.7 mg/m2 orally) on days 1 and 2 (24 hours and 30 minutes before the 5-FU dose) indicated complete oral bioavailability of 5-FU.624 The terminal t1/2 of 5-FU was prolonged to 4.5 hours, and systemic clearance was reduced to 60 mL/min per square meter. The MTD of oral 5-FU was 25 mg/m2 given on days 2 through 6 with eniluracil 3.7 mg/m2orally on days 1 through 7. In another trial, no toxicity was observed after oral eniluracil given at doses of 0.74, 3.7, and 18.5 mg/m2 daily for 7 days.623After a 14-day washout period, eniluracil was given daily for 3 days with 5-FU 10 mg/m2 intravenously on day 2; no toxicity was seen. With 50 mg of eniluracil on days 1 through 3 and either 10 mg/m2 intravenously or 20 mg orally 5-FU on day 2, the t1/2 of 5-FU averaged 4.9 and 6.1 hours, respectively. After a 14-day washout, patients received eniluracil orally on days 1 through 7 with escalating doses of either oral or intravenous 5-FU. Neutropenia and thrombocytopenia were dose-limiting; nonhematologic toxicities (nausea, vomiting, diarrhea, anorexia, mucositis, and fatigue) occurred less frequently. When 5-FU was given on days 2 through 6 with 50 mg each of eniluracil and LV on days 1 through 7, the recommended dose of oral 5-FU was 15 mg/m2, 28-fold lower than the customary dose of 5-FU and LV on a monthly schedule.

A 28-day schedule has been explored in which eniluracil and 5-FU were administered orally twice a day. The recommended doses of eniluracil and 5-FU are 10 and 1 mg/m2 twice daily for 28 of 35 days.625 For the majority of the subsequent phase 2 and 3 trials, a combination tablet that incorporates eniluracil and 5-FU in a dose ratio of 10 to 1 was used. Phase 2 trials indicated the 28-day regimen is active as first-line therapy in breast cancer (52% of 29 patients responded) and colorectal cancer (24% of 45 patients responded).625, 626 In contrast to the experience with CI 5-FU schedules and oral capecitabine, hand-foot syndrome was not observed.

Phase 3 trials that compared the oral 28-day schedule of eniluracil and 5-FU as first-line therapy for colorectal cancer with a daily for 5 days schedule of intravenous bolus 5-FU plus LV failed to demonstrate equivalence for oral eniluracil/5-FU, and clinical development has ceased.627

Some patients who received a subsequent 5-FU-type regimen 3 to 5 weeks after completing protocol therapy with the 28-day schedule of eniluracil and 5-FU experienced life-threatening or fatal toxicity, leading to the recommendation that a minimum of 8 weeks elapse between the last dose of eniluracil and subsequent therapy with another 5-fluoropyrimidine. In a phase 1 trial of oral eniluracil given days 1 to 3 with 5-FU given twice daily on day 2 (intended to simulate a weekly high-dose 24-hour CI schedule), pharmacodynamic studies suggested prolonged inhibition of DPD for up to 19 days after the last dose of eniluracil as reflected by DPD-catalytic activity in PBMCs and elevated uracil levels compared with baseline values.628 Another study evaluated the pharmacodynamic effects of two schedules of eniluracil on a weekly schedule: 20 mg orally on days 1 to 3 with a single dose of 5-FU given day 2, or a single dose of eniluracil and 5-FU. DPD activity was profoundly depressed during oral therapy, and uracil levels were strikingly elevated with both schedules. With the daily-for-3-days schedule, DPD activity was similar to baseline values by 3 weeks after the earlier eniluracil dose, whereas it appeared to recover earlier in patients receiving the single-dose schedule, reaching baseline values by 2 weeks.629 These latter studies raise a question as to whether the dose of eniluracil used in the pivotal studies may have been excessive.

Other Oral 5-Fluoropyrimidines

Several other oral 5-FU prodrugs are either commercially available outside the United States are undergoing clinical investigation.630, 631, 632, 633, 634Carmofur is 5-fluoro-N-hexyl-3,4-dihydro-2,4-dioxopyrimidine-1(2H)-carboxamide (MW 257). S-1 is a three-drug preparation containing ftorafur; 5-chloro-2,4-dihydroxypyridine (CDHP), a competitive, reversible inhibitor of DPD that is about 180-fold more potent than uracil in vitro, and oxonic acid, which strongly inhibits the anabolism of 5-FU to FUMP by OPRTase; the molar ratio is 1.0 to 0.4 to 1.0. BOF-A2 (emitefur, 3-{3-[6-benzoyloxy-3-cyano-2-pyridyloxycarbonyl] benzoyl}-1-ethoxymethyl-5-fluorouracil; MW 558) contains 1-ethoxymethyl-5-fluorouracil, a masked 5-FU prodrug, and 3-cyano-2, 6-dihydroxypyridine (CNDP), a potent inhibitor of DPD. The features of these oral 5-FU prodrugs are highlighted in Table 7-13.

INTERFERENCE OF WARFARIN AND PHENYTOIN METABOLISM BY 5-FLUOROURACIL AND CAPECITABINE

Case reports have described prolongation of the prothrombin time in patients receiving either therapeutic and minidose warfarin in conjunction with 5-FU or capecitabine. In some cases, this effect has been associated with supraanticoagulation and bleeding complications. A retrospective study of 95 patients that employed infusional 5-FU regimens in conjunction with warfarin 1 mg daily to decrease the risk of catheter-associated thrombosis reported elevations of the institutional normalized ratio (INR) of more than 1.5 in 33% of patients; the INR was greater than 3.0 in 19%, and bleeding complications were observed in 8%.635 Similarly, patients receiving phenytoin concurrently with 5-FU have experienced elevated phenytoin concentrations.636 Both warfarin and phenytoin are principally metabolized by cytochrome P-450 2C9. Preclinical studies in rats suggest a probable explanation. Rats treated with a single intraperitoneal dose of 5-FU had decreased protein expression and catalytic activity of two constitutive CYP isozymes, CYP2C11 and CYP3A.637 Rats given oral racemic warfarin during a 8-day intraperitoneal regimen of 5-FU had a significant decrease in the total serum clearance of S-warfarin, which was attributed to a significant decrease in the rate of formation of the oxidative metabolites of the potent S-enantiomer.638 Administration of 5-FU for 7 days reduces phenytoin-p-hydroxylation activity and decreases the total clearance of phenytoin.639 These findings indicate that patients receiving warfarin concurrently with either 5-FU or capecitabine should have their prothrombin time and INR values monitored frequently to allow dose adjustment of warfarin to prevent overanticoagulation.

REFERENCES

1. Heidelberger C, Chaudhuari NK, Daneberg P, et al. Fluorinated pyrimidines. A new class of tumor inhibitory compounds. Nature 1957;179:663–666.

2. Wohlhueter RM, McIvor RS, Plagemann PGW. Facilitated transport of uracil and 5-fluorouracil, and permeation of orotic acid into cultured mammalian cells. J Cell Physiol 1980;104:309–319.

3. Domin BA, Mahony WB, Zimmerman TP. Transport of 5-fluorouracil and uracil into human erythrocytes. Biochem Pharmacol 1993;46:503–510.

4. Pastor-Anglada, M, Felipe A, Casado FJ. Transport and mode of action of nucleoside derivatives used in chemical and antiviral therapies. Trends Pharmacol Sci 1998;19,424–430.

5. Bowen D, Diasio RB, Goldman ID. Distinguishing between membrane transport and intracellular metabolism of fluorodeoxyuridine in Ehrlich ascites tumor cells by application of kinetic and high-performance liquid chromatographic techniques. J Biol Chem 1979;254:5333–5339.

6. Kessel D, Deacon J, Coffey B, et al. Some properties of a pyrimidine phosphoribosyltransferase from murine leukemia cells. Mol Pharmacol 1972;8:731–739.

7. Schwartz PM, Handschumacher RE. Selective antagonism of 5-fluorouracil cytotoxicity by 4-hydroxypyrazolopyrimidine (allopurinol) in vitro. Cancer Res 1979;39:3095–3101.

8. Cory J, Breland JB, Carter GL. Effect of 5-fluorouracil on RNA metabolism in Novikoff hepatoma cells. Cancer Res 1979;39: 4905–4913.

9. Cadman E, Davis L, Heimer R. Enhanced 5-fluorouracil nucleotide formation following methotrexate: biochemical explanation for drug synergism. Science 1979;205:1135–1137.

10. Houghton JA, Houghton RJ. 5-Fluorouracil in combination with hypoxanthine and allopurinol: toxicity and metabolism in xenografts of human colonic carcinomas in mice. Biochem Pharmacol 1980;29:2077–2080.

11. Benz C, Cadman E. Modulation of 5-fluorouracil metabolism and cytotoxicity by antimetabolite pretreatment in human colorectal adenocarcinoma HCT-8. Cancer Res 1981;41:994–999.

12. Houghton JA, Houghton PJ. Elucidation of pathways of 5-fluorouracil metabolism in xenografts of human colorectal adenocarcinoma. Eur J Cancer Clin Oncol 1983;19:807–815.

13. Finan PJ, Kiklitis PA, Chisholm EM, et al. Comparative levels of tissue enzymes concerned in the early metabolism of 5-fluorouracil in normal and malignant human colorectal tissue. Br J Cancer 1984;50:711–715.

14. Schwartz PM, Moir RD, Hyde CM, et al. Role of uridine phosphorylase in the anabolism of 5-fluorouracil. Biochem Pharmacol 1987;34:3585–3589.

15. Woodman PW, Sarrif AM, Heidelberger C. Specificity of pyrimidine nucleoside phosphorylases and the phosphorolysis of 5-fluoro-2′ deoxyuridine. Cancer Res 1980;40:507–511.

16. Niedzwicki JG, El Kouni MH, Chu SH, et al. Structure activity relationship of ligands of the pyrimidine nucleoside phosphorylases. Biochem Pharmacol 1983;32:399–415.

17. Pogolotti AL, Nolan PA, Santi DV. Methods for the complete analysis of 5-fluorouracil metabolites in cell extracts. Anal Biochem 1981;117:178–186.

18. Peterson MS, Ingraham HA, Goulian M. 2′-Deoxyribosyl analogues of UDP-N-acetylglucosamine in cells treated with methotrexate or 5-fluorodeoxyuridine. J Biol Chem 1983;258: 10831–10834.

19. Peters GJ, Laurensse E, Lankelma J, et al. Separation of several 5-fluorouracil metabolites in various melanoma cell lines: evidence for the synthesis of 5-fluorouracil-nucleotide sugars. Eur J Cancer Clin Oncol 1984;20:1425–1431.

20. Santi DV, McHenry CS, Sommer A. Mechanisms of interactions of thymidylate synthetase with 5-fluorodeoxyuridylate. Biochemistry 1974;13:471–480.

21. Sommer A, Santi DV. Purification and amino acid analysis of an active site peptide from thymidylate synthetase containing covalently bound 5′-fluoro-2′-deoxyuridylate and methylene tetrachloride. Biochem Biophys Res Commun 1974;57:689–696.

22. Howell SB, Mansfield SJ, Taetle R. Significance of variation in serum thymidine concentration for the marrow toxicity of methotrexate. Cancer Chemother Pharmacol 1981;5:221–226.

23. Dolnick BJ, Cheng Y-C. Human thymidylate synthetase derived from blast cells of patients with acute myelocytic leukemia. J Biol Chem 1977;252:7697–7703.

24. Dolnick BJ, Cheng Y-C. Human thymidylate synthase: II. Derivatives of pteroylmono- and polyglutamates as substrates and inhibitors. J Biol Chem 1978;253:3563–3567.

25. Fernandes DJ, Bertino JR. 5-Fluorouracil-methotrexate synergy: enhancement of 5-fluorodeoxyuridylate binding to thymidylate synthetase by dihydropteroylpolyglutamates. Proc Natl Acad Sci U S A 1980;77:5663–5667.

26. Allegra CJ, Chabner BA, Jolivet J. Enhanced inhibition of thymidylate synthase by methotrexate polyglutamates. J Biol Chem 1986;230:9720–9726.

27. Ullman B, Lee M, Martin DW Jr, et al. Cytotoxicity of 5- fluoro-2′-deoxyuridine: requirement for reduced folate cofactors and antagonism by methotrexate. Proc Natl Acad U S A 1978; 75:980–983.

28. Danenberg KD, Danenberg PV. Evidence for sequential interaction of the subunits of thymidylate synthetase. J Biol Chem 1979;254:4345–4348.

29. Murinson DS, Anderson T, Schwartz HS, et al. Competitive radioassay for 5-fluorodeoxyuridine 5′-monophosphate in tissues. Cancer Res 1979;39:2471–2479.

21. Washtien WL, Santi DV. Assay of intracellular free and macromolecular-bound metabolites of 5-fluorodeoxyuridine and 5-fluorouracil. Cancer Res 1979;39:3397–3404.

31. Hardy LW, Finer-Moore JS, Montfort WR, et al. Atomic structure of thymidylate synthase: target for rational drug design. Science 1987;235:448–455.

32. Santi DV, McHenry CS, Raines RT, et al. Kinetics and thermodynamics of the interaction of 5-fluouro-2′-deoxyuridylate. Biochemistry 1987;26:8606–8613.

33. Appelt K, Bacquet RJ, Bartlett CA, et al. Design of enzyme inhibitors using iterative protein crystallographic analysis. J Med Chem 1991;34:1925–1934.

34. Schoichet BK, Stroud RM, Santi DV, et al. Structure-based discovery of inhibitors of thymidylate synthase. Science 1993;259: 1445–1450.

35. Maybaum J, Ullman B, Mandel HG, et al. Regulation of RNA- and DNA-directed actions of 5-fluoropyrimidines in mouse T-lymphoma (S-49) cells. Cancer Res 1980;40:4209–4215.

36. Evans RM, Laskin JD, Hakala MT. Assessment of growth-limiting events caused by 5-fluorouracil in mouse cells and in human cells. Cancer Res 1980;40:4113–4122.

37. Spiegelman S, Sawyer R, Nayak R, et al. Improving the antitumor activity of 5-fluorouracil by increasing its incorporation into RNA via metabolic modulation. Proc Natl Acad Sci U S A 1980;77:4996–4970.

38. Santelli G, Valeriote F. In vivo enhancement of 5-fluorouracil cytotoxicity to AKR leukemia cells by thymidine in mice. J Natl Cancer Inst 1978;61:843–847.

39. Carrico CK, Glazer RI. Augmentation by thymidine of the incorporation and distribution of 5-fluorouracil into ribosomal RNA. Biochem Biophys Res Commun 1979;87:664–670.

40. Kufe DW, Egan EM. Enhancement of 5-fluorouracil incorporation into human lymphoblast ribonucleic acid. Biochem Pharmacol 1981;30:129–133.

41. Wilkinson DS, Tisty TD, Hanas RJ. The inhibition of ribosomal RNA synthesis and maturation in Novikoff hepatoma cells by 5-fluorouridine. Cancer Res 1975;35: 3014–3020.

42. Chaudhuri NK, Montag BJ, Heidelberger C. Studies on fluorinated pyrimidines: III. The metabolism of 5-fluorouracil-2- (14C and 5-fluoroorotic-2- (14C acid in vivo. Cancer Res 1958;18:318–328.

43. Herrick D, Kufe DW. Lethality associated with incorporation of 5-fluorouracil into preribosomal RNA. Mol Pharmacol 1984;26:135–140.

44. Kanamaru R, Kakuta H, Sato T, et al. The inhibitory effects of 5-fluorouracil on the metabolism of preribosomal and ribosomal RNA in L-1210 cells in vitro. Cancer Chemother Pharmacol 1986;17:43–46.

45. Greenhalgh DA, Parish JH. Effect of 5-fluorouracil combination therapy on RNA processing in human colonic carcinoma cells. Br J Cancer 1990;61:415–419.

46. Ghoshal K, Jacob ST. Specific inhibition of pre-ribosomal RNA processing in extracts from the lymphosarcoma cells treated with 5-fluorouracil. Cancer Res 1994;54:632–636.

47. Ghoshal K, Jacob ST. An alternative molecular mechanism of action of 5-fluorouracil, a potent anticancer drug. Biochem Pharmacol 1997;53:1569–1575.

48. Kufe DW, Major PP. 5-Fluorouracil incorporation into human breast carcinoma RNA correlates with cytotoxicity. J Biol Chem 1981;256:9802–9805.

49. Glazer RI, Lloyd LS. Association of cell lethality with incorporation of 5-fluorouracil and 5-fluorouridine into nuclear RNA in human colon carcinoma cells in culture. Mol Pharmacol 1982;21:468–473.

50. Laskin JD, Evans RM, Slocum HK, et al. Basis for natural variation in sensitivity to 5-fluorouracil in mouse and human cells in culture. Cancer Res 1979;39:383–390.

51. Spears CP, Shani J, Shahinian AH, et al. Assay and time course of 5-fluorouracil incorporation into RNA of L1210/ 0 ascites cells in vivo. Mol Pharmacol 1985;27:302–307.

52. Carrico CK, Glazer RI. The effect of 5-fluorouracil on the synthesis and translation of poly(A) RNA from regenerating liver. Cancer Res 1979;39:3694–3701.

53. Tseng W-C, Medina D, Randerath K. Specific inhibition of transfer RNA methylation and modification in tissue of mice treated with 5-fluorouracil. Cancer Res 1978;38:1250–1257.

54. Will CL, Dolnick BJ. 5-Fluorouracil inhibits dihydrofolate reductase precursor mRNA processing and/or nuclear mRNA stability in methotrexate-resistant KB cells. J Biol Chem 1989;264:21413–21421.

55. Iwata T, Watanabe T, Kufe DW. Effects of 5-fluorouracil on globin mRNA synthesis in murine erythroleukemia cells. Biochemistry 1986;25:2703–2707.

56. Armstrong RD, Lewis M, Stern SG, et al. Acute effect of 5-fluorouracil on cytoplasmic and nuclear dihydrofolate reductase messenger RNA metabolism. J Biol Chem 1986;261:7366–7371.

57. Armstrong RD. RNA as a target for antimetabolites. In: Glazer RI, ed. Developments in Cancer Chemotherapy. Vol 2. Boca Raton, FL: CRC Press, 1989:154–174.

58. Takimoto CH, Voeller DB, Strong JM, et al. Effects of 5-fluorouracil substitution on the RNA conformation and in vitro translation of thymidylate synthase messenger RNA. J Biol Chem 1993;28:21438–21442.

59. Schmittgen TD, Danenberg KD, Horikoshi T, et al. Effect of 5-fluoro- and 5-bromouracil substitution on the translation of human thymidylate synthase mRNA. J Biol Chem 1994;269: 16269–16275.

60. Armstrong RD, Takimoto CH, Cadman EC. Fluoropyrimidine-mediated changes in small nuclear RNA. J Biol Chem 1986; 261:21–24.

61. Takimoto CH, Cadman EC, Armstrong RD. Precursor- dependent differences in the incorporation of fluorouracil in RNA. Mol Pharmacol 1986;29:637–642.

62. Sierakowska H, Shukla RR, Dominsksi A, et al. Inhibition of pre-mRNA splicing by 5-fluoro-, 5-chloro- and 5-bromouridine. J Biol Chem 1989;264:19185–19191.

63. Doong SL, Dolnick BJ. 5-Fluorouracil substitution alters pre-mRNA splicing in vitro. J Biol Chem 1988;263:4467–4473.

64. Danenberg PV, Shea LCC, Danenberg K. Effect of 5-fluorouracil substitution on the self-splicing activity of Tetrahymena ribosomal RNA. Cancer Res 1990;50:1757–1763.

65. Lenz H-J, Manno DJ, Danenberg KD, et al. Incorporation of 5-fluorouracil into U2 and U6 snRNA inhibits mRNA precursor splicing. J Biol Chem 1994;269:31962–31968.

66. Randerath K, Tseng W-C, Harris JS, et al. Specific effects of fluoropyrimidines and 5-azapyrimidines on modification of the 5 position of pyrimidines, in particular the synthesis of 5-methyluracil and 5-methylcytosine in nucleic acids. Cancer Res 1983;84:283–297.

67. Santi DV, Hardy LW. Catalytic mechanism and inhibition of tRNA (uracil-5-)methyltransferase: evidence for covalent catalysis. Biochemistry 1987;26:8599–8606.

68. Samuelsson T. Interactions of transfer RNA pseudouridine synthases with RNAs substituted with fluorouracil. Nucleic Acids Res 1991;19:6139–6144.

69. Patton JR. Ribonucleoprotein particle assembly and modification of U2 small nuclear RNA containing 5-fluorouridine. Biochemistry 1993;32:8939–9844.

70. Shani J, Danenberg PV. Evidence that intracellular synthesis of 5-fluorouridine-5′-phosphate from 5-fluorouracil and 5- fluorouridine is compartmentalized. Biochem Biophys Res Commun 1984;122:439–445.

71. Jin Y, Heck DE, DeGeorge G, et al. 5-Fluorouracil suppresses nitric oxide biosynthesis in colon carcinoma cells. Cancer Res 1996;56:1978–1982.

72. Fujishima H, Niho Y, Kondo T, et al. Inhibition by 5-fluorouracil of ERCC1 and gamma-glutamylcysteine synthetase messenger RNA expression in a cisplatin-resistant HST-1 human squamous carcinoma cell line. Oncol Res 1997;9:167–172.

73. Grem JL, Mulcahy RT, Miller EM, et al. Interaction of deoxyuridine with fluorouracil and dipyridamole in a human colon cancer cell line. Biochem Pharmacol 1989;38:51–59.

74. Curtin NJ, Harris AL, Aherne GW. Mechanism of cell death following thymidylate synthase inhibition: 2′-deoxy- 5′-triphosphate accumulation, DNA damage, and growth inhibition following exposure to CB3717 and dipyridamole. Cancer Res 1991;51:2346–2352.

75. Aherne GW, Hardcastle A, Raynaud F, et al. Immunoreactive dUMP and TTP pools as an index of thymidylate synthase inhibition; effect of tomudex (ZD1694) and a nonpolyglutamated quinazoline antifolate (CB30900) in L1210 mouse leukaemia cells. Biochem Pharmacol 1996;51:1293–1301.

76. Tanaka M, Yoshida S, Saneyoshi M, et al. Utilization of 5- fluoro-2′-deoxyuridine triphosphate and 5-fluoro-2′-deoxycytidine triphosphate in DNA synthesis by DNA polymerases alpha and beta from calf thymus. Cancer Res 1981;41: 4132–4135.

77. Ingraham HA, Tseng BY, Goulian M. Mechanism for exclusion of 5-fluorouracil from DNA. Cancer Res 1980;40:998–1001.

78. Herrick D, Major PP, Kufe DW. Effect of methotrexate on incorporation and excision of 5-fluorouracil residues in human breast carcinoma DNA. Cancer Res 1982;42:5015–5017.

79. Cheng Y-C, Nakayama K. Effects of 5-fluoro-2′-deoxyuridine on DNA metabolism in HeLa cells. Mol Pharmacol 1983;23: 171–174.

80. Tanaka M, Kimura K, Yoshida S. Enhancement of the incorporation of 5-fluorodeoxyuridylate into DNA of HL-60 cells by metabolic modulations. Cancer Res 1983;43:5145–5150.

81. Kufe DW, Scott P, Fram R, et al. Biologic effect of 5-fluoro- 2′-deoxyuridine incorporation in L1210 deoxyribonucleic acid. Biochem Pharmacol 1983;32:1337–1340.

82. Schuetz JD, Wallace HJ, Diasio RB. 5-Fluorouracil incorporation into DNA of CF-1 mouse bone marrow cells as a possible mechanism of toxicity. Cancer Res 1984;44:1358–1363.

83. Sawyer RC, Stolfi RL, Martin DS, et al. Incorporation of 5- fluorouracil into murine bone marrow DNA in vivo. Cancer Res 1984;44:1847–1851.

84. Caradonna DJ, Cheng YC. The role of deoxyuridine triphosphate nucleotidohydrolase, uracil-DNA glycosylase, and DNA polymerase alpha in the metabolism of FUdR in human tumor cells. Mol Pharmacol 1980;18:513–520.

85. Chu E, Lai GM, Zinn S, et al. Resistance of a human ovarian cancer line to 5-fluorouracil associated with decreased levels of 5-fluorouracil in DNA. Mol Pharmacol 1990;38:410–417.

86. Harris JM, McIntosh EM, Muscat GE. Structure/function analysis of a dUTPase: catalytic mechanism of a potential chemotherapeutic target. J Mol Biol 1999;2:275–287.

87. Canman CE, Lawrence TS, Shewach DS, et al. Resistance to fluorodeoxyuridine-induced DNA damage and cytotoxicity correlates with an elevation of deoxyuridine triphosphatase activity and failure to accumulate deoxyuridine triphosphate. Cancer Res 1993;53:5219–5224.

88. Mauro DJ, De Riel JK, Tallarida RJ, et al. Mechanisms of excision of 5-fluorouracil by uracil DNA glycosylase in normal human cells. Mol Pharmacol 1993;43:854–857.

89. Wurzer JC, Tallarida RJ, Sirover MA. New mechanism of action of the cancer chemotherapeutic agent 5-fluorouracil in human cells. J Pharmacol Exp Ther 1994;269:39–43.

90. Yoshioka A, Tanaka S, Hiraoka O, et al. Deoxyribonucleoside triphosphate imbalance—fluorodeoxyuridine-induced DNA double strand breaks in mouse FM3A cells and the mechanism of cell death. J Biol Chem 1987;262:8235–8241.

91. Houghton JA, Tillman DM, Harwood FG. Ratio of 2′- deoxyadenosine-5′-triphosphate/thymidine-5′-triphosphate influences the commitment of human colon carcinoma cells to thymineless death. Clin Cancer Res 1995;1:723–730.

92. Wadler S, Horowitz R, Mao X, et al. Effect of interferon of 5-fluorouracil-induced perturbations in pools of deoxynucleotide triphosphates and DNA strand breaks. Cancer Chemother Pharmacol 1996;38:529–535.

93. Schuetz JD, Collins JM, Wallace HJ, et al. Alteration of the secondary structure of newly synthesized DNA from murine bone marrow cells by 5-fluorouracil. Cancer Res 1986;46:119–123.

94. Jones S, Willmore E, Durkacz BW. The effects of 5-fluoropyrimidines on nascent DNA synthesis in Chinese hamster ovary cells monitored by pH-step alkaline and neutral elution. Carcinogenesis 1994;15:2435–2438.

95. Yin M, Rustum YM. Comparative DNA strand breakage induced by FUra and FdUrd in human ileocecal adenocarcinoma (HCT-8) cells: relevance to cell growth inhibition. Cancer Commun 1991;3:45–51.

96. Lonn U, Lonn S. Increased levels of DNA lesions induced by leucovorin-5-fluoropyrimidine in human colon adenocarcinoma. Cancer Res 1988;48:4153–4157.

97. Dusenbury CE, Davis MA, Lawrence TS, et al. Induction of megabase DNA fragments by 5-fluorodeoxyuridine in human colorectal tumor (HT29) cells. Mol Pharmacol 1991;39:285–289.

98. Canman CE, Tang H-Y, Normolle DP, et al. Variations in patterns of DNA damage induced in human colorectal tumor cells by 5-fluorodeoxyuridine. Implications for mechanisms of resistance and cytotoxicity. Proc Natl Acad U S A 1992;89:10474–10478.

99. Ayusawa D, Arai H, Wataya Y, et al. A specialized form of chromosomal DNA degradation induced by thymidylate stress in mouse FM3A cells. Mutat Res 1988;200:221–230.

100. Li Z-R, Yin M-B, Arredendo MA, et al. Down-regulation of c-myc gene expression with induction of high molecular weight DNA fragments by fluorodeoxyuridine. Biochem Pharmacol 1994;48:327–334.

101. Kyprianou N, Isaacs JT. “Thymineless” death in androgen- independent prostatic cancer cells. Biochem Biophys Res Commun 1989;165:73–81.

102. Lowe SW, Ruley HE, Jacks T, et al. p53-Dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 1993;74:957–967.

103. Fisher TC, Milner AE, Gregory CD, et al. Bcl-2 modulation of apoptosis induced by anticancer drugs: resistance to thymidylate stress is independent of classical resistance pathways. Cancer Res 1993;53:3321–3326.

104. Lonn U, Lonn S. The increased cytotoxicity in colon adenocarcinoma of methotrexate-5-fluorouracil is not associated with increased induction of lesions in DNA by 5-fluorouracil. Biochem Pharmacol 1986;35:177–181.

105. Parker WB, Kennedy KA, Klubes P. Dissociation of 5-fluorouracil- induced DNA fragmentation from either its incorporation into DNA or its cytotoxicity in murine T- lymphoma (S-49). Cancer Res 1987;47:979–982.

106. Darzynkiewicz Z. Methods in analysis of apoptosis and cell necrosis. In: Parker J, Stewart C, eds. The Purdue Cytometry CD-ROM. Vol 3. West Lafayette, IN: Purdue University, 1997.

107. Houghton JA, Harwood FG, Tillman DM. Thymineless death in colon carcinoma cells is mediated via Fas signaling. Proc Natl Acad U S A 1997;94:8144–8149.

108. Tillman DM, Petak I, Houghton JA. A fas-dependent component in 5-fluorouracil/leucovorin-induced cytotoxicity in colon carcinoma cells. Clin Cancer Res 1999;5:425–430.

109. Ciccolini J, Peillard L, Evrard A, et al. Enhanced antitumor activity of 5-fluorouracil in combination with 2′-deoxyinosine in human colorectal cell lines and human colon tumor xenografts. Clin Cancer Res 2000;61529-35.

110. Longley DB, Allen WL, McDermott U, et al. The roles of thymidylate synthase and p53 in regulating Fas-mediated apoptosis in response to antimetabolites. Clin Cancer Res 2004;10:3562-3571.

111. Pritchard DM, Watson AJM, Potten CS, et al. Inhibition of uridine but not thymidine of p53-dependent intestinal apoptosis initiated by 5-fluorouracil: evidence for the involvement of RNA perturbation. Proc Natl Acad U S A 1997;94:1795–1799.

112. Li L, Berger SH, Wyatt MD. Involvement of base excision repair in response to therapy targeted at thymidylate synthase. Mol Cancer Ther 2004;3(6):747-53.

113. Goel A, Arnold CN, Boland CR: Multistep progression of colorectal cancer in the setting of microsatellite instability: new details and novel insights. Gastroenterology 121:1497-502, 2001.

114. Meyers M, Wagner MW, Hwang HS, et al. Role of the hMLH1 DNA mismatch repair protein in fluoropyrimidine-mediated cell death and cell cycle responses. Cancer Res 2001;61(13): 5193-201.

115. Arnold CN, Goel A, Boland CR. Role of hMLH1 promoter hypermethylation in drug resistance to 5-fluorouracil in colorectal cancer cell lines. Int J Cancer 2003;106(1):66-73.

116. Gryfe R, Kim H, Hsieh ET, et al. Tumor microsatellite instability and clinical outcome in young patients with colorectal cancer. N Engl J Med 342:69-77, 2000

117. Aschele C, Sobrero A, Faderan MA, et al. Novel mechanisms of resistance to 5-fluorouracil in human colon cancer (HCT- 8) sublines following exposure to two different clinically relevant dose schedules. Cancer Res 1992;52:1855–1964.

118. Sobrero AF, Aschele C, Guglielmi AP, et al. Synergism and lack of cross-resistance between short-term and continuous exposure to fluorouracil in human colon adenocarcinoma cells. J Natl Cancer Inst 1993;85:1937–1944.

119. Ren Q-F, Van Groeningen CJ, Geoffroy F, et al. Determinants of cytotoxicity with prolonged exposure to fluorouracil in human colon cancer cells. Oncol Res 1997;9:77–88.

120. Reichard P, Skold O, Klein G, et al. Studies on resistance against 5-fluorouracil: I. Enzymes of the uracil pathway during development of resistance. Cancer Res 1962;22:235–243.

121. Ardalan B, Cooney DA, Jayaram HN, et al. Mechanisms of sensitivity and resistance of murine tumors to 5-fluorouracil. Cancer Res 1980;40:1431–1437.

122. Mulkins MA, Heidelberger C. Isolation of fluoropyrimidine-resistant murine leukemic cell lines by one-step mutation and selection. Cancer Res 1982;42:956–964.

123. Mulkins MA, Heidelberger C. Biochemical characterization of fluoropyrimidine-resistant murine leukemic cell lines. Cancer Res 1982;42:965–973.

124. Piper AA, Fox RM. Biochemical basis for the differential sensitivity of human T- and B-lymphocyte lines to 5-fluorouracil. Cancer Res 1982;42:3753–3760.

125. Ardalan B, Villacorte D, Heck D, et al. Phosphoribosyl pyrophosphate pool size and tissue levels as a determinant of 5-fluorouracil response in murine colonic adenocarcinomas. Biochem Pharmacol 1982;31:1989–1992.

126. Au J L-S, Rustum YM, Minowad J, et al. Differential selectivity of 5-fluorouracil and 5′-deoxy-5-fluorouridine in cultured human B lymphocytes and mouse L1210 leukemia. Biochem Pharmacol 1983;32:541–546.

127. Yoshida M, Hoshi A. Mechanism of inhibition of phosphoribosylation of 5-fluorouracil by purines. Biochem Pharmacol 1984;33:2863–2867.

128. El-Assouli SM. The molecular basis for the differential sensitivity of B and T lymphocytes to growth inhibition by thymidine and 5-fluorouracil. Leuk Res 1985;9:391–398.

129. Peters GJ, Laurensse E, Leyva A, et al. Sensitivity of human, murine and rat cells to 5-fluorouracil and 5′-deoxy-5-fluorouridine in relation to drug-metabolizing enzymes. Cancer Res 1986;46:20–28.

130. Tamemasa O, Tezuka M. Additive formation of antineoplastic 5-fluorouracil nucleosides from 5-fluorouracil by Ehrlich ascites tumor extracts in the presence of ribose 1- phosphate/uridine or deoxyribose 1-phosphate/deoxyuridine. J Pharmacobiodyn 1982;5:720–726.

131. Beltz RE, Waters RN, Hegarty TJ. Enhancement and depression by inosine of the growth inhibitory action of 5- fluorouracil on cultured Jensen tumor cells. Biochem Biophys Res Commun 1983;112:235–241.

132. Washtien WL. Comparison of 5-fluorouracil metabolism in two human gastrointestinal tumor cell lines. Cancer Res 1984;44:909–914.

133. Iigo M, Yamaizumi Z, Nishimura S, et al. Mechanism of potentiation of antitumor activity of 5-fluorouracil by guanine ribonucleotides against adenocarcinoma 755. Eur J Cancer Clin Oncol 1987;23:1059–1065.

134. Klubes P, Connelly K, Cerna I, et al. Effects of 5-fluorouracil on 5-fluorodeoxyuridine 5-monophosphate and 2- deoxyuridine 5′-monophosphate pools and DNA synthesis in solid mouse L1210 and rat Walker 256 tumors. Cancer Res 1978;38: 2325–2331.

135. Fernandes DJ, Cranford SK. Resistance of CCRF-CEM cloned sublines to 5-fluorodeoxyuridine associated with enhanced phosphatase activities. Biochem Pharmacol 1985;34: 125–132.

136. Moran RG, Spears CP, Heidelberger C. Biochemical determinants of tumor sensitivity to 5-fluorouracil: ultrasensitive methods for determination of 5-fluoro-2′-deoxyuridylate, 2′-deoxyuridylate, and thymidylate synthetase. Proc Natl Acad Sci U S A 1979;76:1456–1460.

137. Berger SH, Hakala MT. Relationship of dUMP and free FdUMP pools to inhibition to thymidylate synthase by 5- fluorouracil. Mol Pharmacol 1984;25:303–309.

138. Houghton JA, Weiss KD, Williams LG, et al. Relationship between 5-fluoro-2′-deoxyuridylate, 2′-deoxyuridylate, and thymidylate synthase activity subsequent to 5-fluorouracil administration, in xenografts of human colon adenocarcinomas. Biochem Pharmacol 1986;35:1351–1358.

139. Kaufman ER. Resistance to 5-fluorouracil associated with increased cytidine triphosphate levels in V79 Chinese hamster cells. Cancer Res 1984;44:3371–3376.

140. Aronow B, Watts T, Lassetter J, et al. Biochemical phenotype of 5-fluorouracil-resistant murine T-lymphoblasts with genetically altered CTP synthetase activity. J Biol Chem 1984;259: 9035–9043.

141. Noordhuis P, Holwerda U, Van Der Wilt CL, et al. 5-Fluorouracil incorporation into RNA and DNA in relation to thymidylate synthase inhibition of human colorectal cancers. Ann Oncol 2004;15(7):1025-32.

142. Fernandes DJ, Cranford SK. A method for the determination of total, free, and 5-fluorodeoxyuridylate-bound thymidylate synthase in cell extracts. Anal Biochem 1984;142:378–385.

143. Yalowich JC, Kalman TI. Rapid determinations of thymidylate synthase activity and its inhibition in intact L1210 leukemia cells in vitro. Biochem Pharmacol 1985;34:2319–2324.

144. Spears CP, Gustavsson BG, Mitchell MS, et al. Thymidylate synthetase inhibition in malignant tumors and normal liver of patients given intravenous 5-fluorouracil. Cancer Res 1984; 44:4144–4150.

145. Peters GJ, van Groeningen CJ, Leurensse EJ, et al. Thymidylate synthase from untreated human colorectal cancer and colonic mucosa: enzyme activity and inhibition by 5-fluoro-2-deoxyuridine-5-monophosphate. Eur J Cancer 1991;27:263–267.

146. Horikoshi T, Danenberg KD, Staglbauer THW, et al. Quantitation of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase gene expression in human tumors using the polymerase chain reaction. Cancer Res 1992;52:108–116.

147. Lenz H-J, Leichman CG, Danenberg KD, et al. Thymidylate synthase mRNA level in adenocarcinoma of the stomach: a predictor for primary tumor response and overall survival. J Clin Oncol 1985;14:176–182.

148. Leichman CG, Lenz H-J, Leichman L, et al. Quantitation of intratumoral thymidylate synthase expression predicts for disseminated colorectal cancer response and resistance to protracted-infusion fluorouracil and weekly leucovorin. J Clin Oncol 1997;15:3223–3229.

149. Johnston PG, Liang C-M, Henry S, et al. Production and characterization of monoclonal antibodies that localize human thymidylate synthase in the cytoplasm of human cells and tissue. Cancer Res 1991;51:6668–6676.

150. Johnston PG, Drake JC, Trepel J, et al. Immunological quantitation of thymidylate synthase using the monoclonal antibody TS 106 in 5-fluorouracil-sensitive and -resistant human cancer cell lines. Cancer Res 1992;52:4306–4312.

151. Johnston PG, Drake JC, Steinberg SM, et al. The quantitation of thymidylate synthase in human tumors using an ultrasensitive enzyme-linked immunoassay. Biochem Pharmacol 1993;12: 2483–2486.

152. Popat S, Matakidou A, Houlston RS. Thymidylate synthase expression and prognosis in colorectal cancer: a systematic review and meta-analysis. J Clin Oncol 2004;22(3):529–536

153. Berger SH, Jenh C-H, Johnson LF, et al. Thymidylate synthase overproduction and gene amplification in fluorodeoxyuridine- resistant human cells. Mol Pharmacol 1985;28:461–467.

154. Clark JL, Berger SH, Mittelman A, et al. Thymidylate synthase gene amplification in a colon tumor resistant to fluoropyrimidine chemotherapy. Cancer Treat Rep 1987;71:261–265.

155. Copur S, Aiba K, Drake JC, et al. Thymidylate synthase gene amplification in human colon cancer cell lines resistant to 5-fluorouracil. Biochem Pharmacol 1995;49:1419–1426.

156. Jastreboff MM, Kedzierska B, Rode W. Altered thymidylate synthetase in 5-fluorodeoxyuridine-resistant Ehrlich ascites carcinoma cells. Biochem Pharmacol 1985;32:2259–2267.

157. Bapat AR, Zarow C, Danenberg PV. Human leukemic cells resistant to 5-fluoro-2′deoxyuridine contain a thymidylate synthase with a lower affinity for nucleotides. J Biol Chem 1983; 258:4130–4136.

158. Berger SH, Barbour KW, Berger FG. A naturally occurring variation in thymidylate synthase structure is associated with a reduced response to 5-fluoro-2′-deoxyuridine in a human colon tumor cell line. Mol Pharmacol 1988;34:480–484.

159. Barbour KW, Berger SH, Berger SG. Single amino acid substitution defines a naturally occurring genetic variant of human thymidylate synthase. Mol Pharmacol 1990;37:515–518.

160. Kawate H, Landis DM, Loeb LA. Distribution of mutations in human thymidylate synthase yielding resistance to 5-fluorodeoxyuridine. J Biol Chem 2002;277(39):36304–36311.

161. Lu K, McGuire JJ, Slocum HK, et al. Mechanisms of acquired resistance to modulation of 5-fluorouracil by leucovorin in HCT-8 human ileocecal carcinoma cells. Biochem Pharmacol 1997;53:689–696.

162. Grem JL, Hoth DF, Hamilton JM, et al. Overview of current status and future direction of clinical trials with 5-fluorouracil in combination with folinic acid. Cancer Treat Rep 1987;71:1249–1264.

163. Zhang Z-G, Rustum YM. Effects of diastereoisomers of 5- formyl-tetrahydrofolate on cellular growth, sensitivity to 5-fluoro-2′-deoxyuridine, and methylenetetrahydrofolate polyglutamate levels in HCT-8 cells. Cancer Res 1991; 51:3476–3481.

164. Boarman DM, Allegra CJ. Intracellular metabolism of 5- formyltetrahydrofolate in human breast and colon cell lines. Cancer Res 1992;52:36–44.

165. Romanini A, Lin JT, Niedzwiecki D, et al. Role of folylpolyglutamates in biochemical modulation of fluoropyrimidines by leucovorin. Cancer Res 1991;51:789–793.

166. Wang F-S, Aschele C, Sobrero A, et al. Decreased folylpolyglutamate synthetase expression: a novel mechanism of fluorouracil resistance. Cancer Res 1993;53:3677–3680.

167. Cohen A, Ullman B. Role of intracellular dTTP levels in fluorodeoxyuridine toxicity. Biochem Pharmacol 1984;33: 3298–3301.

168. Grem JL, Fischer PH. Enhancement of 5-fluorouracil's anticancer activity by dipyridamole. Pharmacol Ther 1989;40:349–371.

169. Radparvar S, Houghton PJ, Germain G, et al. Cellular pharmacology of 5-fluorouracil in a human colon adenocarcinoma cell line selected for thymidine kinase deficiency. Biochem Pharmacol 1990;39:1759–1765.

170. Sobrero AF, Moir RD, Bertino JR, et al. Defective facilitated diffusion of nucleosides, a primary mechanism of resistance to 5-fluoro-2′-deoxyuridine in the HCT-8 human carcinoma. Cancer Res 1985;45:3155–3160.

171. Sobrero AF, Handschumacher RE, Bertino JR. Highly selective drug combinations for human colon cancer cells resistant in vitro to 5-fluoro-2′-deoxyuridine. Cancer Res 1985;45:3161–3163.

172. Jenh C-H, Rao LG, Johnson LF. Regulation of thymidylate synthase enzyme synthesis in 5-fluorodeoxyuridine-resistant mouse fibroblasts during the transition from the resting to growing state. J Cell Physiol 1985;122:149–154.

173. Cadman E, Heimer R. Levels of thymidylate synthetase during normal culture growth of L1210 cells. Cancer Res 1986;46: 1195–1198.

174. Johnson LF. Post transcriptional regulation of thymidylate synthase gene expression. J Cell Biochem 1994;54:378–392.

175. Horie N, Takeishi K. Identification of functional elements in the promoter region of the human gene for thymidylate synthase and nuclear factors that regulate the expression of the gene. J Biol Chem 1997;272:18375–18381.

176. Berne M, Gustavsson B, Almersjo O, et al. Concurrent allopurinol and 5-fluorouracil: 5-fluoro-2′-deoxyuridylate formation and thymidylate synthase inhibition in rat colon carcinoma in regenerating rat liver. Cancer Chemother Pharmacol 1987;20: 193–197.

177. Chu E, Zinn S, Boarman D, et al. Interaction of interferon and 5-fluorouracil in the H630 human colon carcinoma cell line. Cancer Res 1990;50:5834–5840.

178. Van der Wilt CL, Pinedo HM, Smid K, et al. Elevation of thymidylate synthase following 5-fluorouracil treatment is prevented by the addition of leucovorin in murine colon tumors. Cancer Res 1992;52:4922–4928.

179. Parr AL, Drake JC, Gress RE, et al. 5-Fluorouracil-mediated thymidylate synthase induction in malignant and nonmalignant human cells. Biochem Pharmacol 1998;56:231–235.

180. Swain SM, Lippman ME, Chabner BA, et al. Fluorouracil and high-dose leucovorin in previously treated patients with metastatic breast cancer. J Clin Oncol 1989;7:890–899.

181. Chu E, Voeller DM, Johnston PG, et al. Regulation of thymidylate synthase in human colon cancer cells treated with 5-fluorouracil and interferon-gamma. Mol Pharmacol 1993;43: 527–533.

182. Chu E, Koeller DM, Casey JL, et al. Autoregulation of human thymidylate synthase messenger RNA translation by thymidylate synthase. Proc Natl Acad U S A 1991;88:8977–8981.

183. Chu E, Voeller D, Koeller DM, et al. Identification of an RNA binding site for human thymidylate synthase. Proc Natl Acad USA 1993;90:517–521.

184. Ju J, Kane SE, Lenz HJ, et al. Desensitization and sensitization of cells to fluoropyrimidines with different antisenses directed against thymidylate synthase messenger RNA. Clin Cancer Res 1998;4:2229–2236.

185. Schmitz JC, Chen TM, Chu E. Small interfering double-stranded RNAs as therapeutic molecules to restore chemosensitivity to thymidylate synthase inhibitor compounds. Cancer Res 2004;64:1431–1435.

186. Houghton PJ, Germain GS, Hazelton VJ, et al. Mutant of human colon adenocarcinoma selected for thymidylate synthase deficiency. Proc Natl Acad Sci U S A 1989;86:1377–1381.

187. Houghton PJ, Rahman A, Will CL, et al. Mutations of the thymidylate synthase gene of human adenocarcinoma cells causes a thymidylate synthase-negative phenotype that can be attenuated by exogenous folates. Cancer Res 1992;52:558–565.

188. Calabro-Jones PM, Byfield JE, Ward JF, et al. Time-dose relationships for 5-fluorouracil cytotoxicity against human epithelial cancer cells in vitro. Cancer Res 1982;42:4413–4420.

189. Santelli G, Valeriote F. Schedule-dependent cytotoxicity of 5-fluorouracil in mice. J Natl Cancer Inst 1986;76:159–164.

190. Moran RG, Scanlon KL. Schedule-dependent enhancement of the cytotoxicity of fluoropyrimidines to human carcinoma cells in the presence of folinic acid. Cancer Res 1991;51:4618–4623.

191. van Gröeningen CJ, Pinedo HM, Heddes J, et al. Pharmacokinetics of 5-fluorouracil assessed with a sensitive mass spectrometric method in patients on a dose escalation schedule. Cancer Res 1988;48:6956–6961.

192. Anderson LW, Parker RJ, Collins JM, et al. Gas chromatographic-mass spectrometric method for routine monitoring of 5-fluorouracil in plasma of patients receiving low-level protracted infusions. J Chromatogr 1992;581:195–201.

193. Martino R, Malet-Martino M, Gilarad V. Fluorine nuclear magnetic resonance, a privileged tool for metabolic studies of fluoropyrimidine drugs. Curr Drug Metab 2000;1:271–303.

194. Murphy RF, Balis FM, Poplack DG. Stability of 5-fluorouracil in whole blood and plasma. Clin Chem 1987;33:2299–2300.

195. Almersjo OE, Gustavsson BG, Regardh CG, et al. Pharmacokinetic studies of 5-fluorouracil after oral and intravenous administration in man. Acta Pharmacol Toxicol 1980;46:329–336.

196. Christophidis N, Vajda FJE, Lucas I, et al. Fluorouracil therapy in patients with carcinoma of the large bowel: a pharmacokinetic comparison of various rates and routes of administration. Clin Pharmacokinet 1978;3:330–336.

197. Fraile RJ, Baker LH, Buroker TR, et al. Pharmacokinetics of 5-fluorouracil administered orally by rapid intravenous and by slow infusion. Cancer Res 1980;40:2223–2228.

198. Grem JL, McAtee N, Murphy RF, et al. A pilot study of interferon alfa-2a in combination with fluorouracil plus high-dose leucovorin in metastatic gastrointestinal carcinoma. J Clin Oncol 1991;9:1811–1820.

199. MacMillan WE, Wolberg WH, Welling PG. Pharmacokinetics of fluorouracil in humans. Cancer Res 1978;38:3479–3482.

200. Heggie GD, Sommadossi J-P, Cross DS, et al. Clinical pharmacokinetics of 5-fluorouracil and its metabolites in plasma, urine, and bile. Cancer Res 1987;47:2203–2206.

201. Grem JL, McAtee N, Murphy RF, et al. Phase I and pharmacokinetic study of recombinant human granulocyte-macrophage colony-stimulating factor given in combination with fluorouracil plus calcium leucovorin in metastatic gastrointestinal adenocarcinoma. J Clin Oncol 1994;12:560–568.

202. McDermott BJ, van der Berg HW, Murphy RF. Nonlinear pharmacokinetics for the elimination of 5-fluorouracil after intravenous administration in cancer patients. Cancer Chemother Pharmacol 1982;9:173–178.

203. Benz C, DeGregorio M, Saks S, et al. Sequential infusions of methotrexate and 5-fluorouracil in advanced cancer: pharmacology, toxicity, and response. Cancer Res 1985;45:3354–3358.

204. Yoshida T, Araki E, Iigo M, et al. Clinical significance of monitoring serum levels of 5-fluorouracil by continuous infusion in patients with advanced colonic cancer. Cancer Chemother Pharmacol 1990;26:352–354.

205. Harris BE, Song R, Soong SJ, et al. Relationship between dihydropyrimidine dehydrogenase activity and plasma 5-fluorouracil levels with evidence for circadian variation of enzyme activity and plasma drug levels in cancer patients receiving 5-fluorouracil by protracted continuous infusion. Cancer Res 1990;50:197–201.

206. Grem JL, McAtee N, Balis F, et al. A phase II study of continuous infusion 5-fluorouracil and leucovorin with weekly cisplatin in metastatic colorectal carcinoma. Cancer 1993; 72:663–668.

207. Petit E, Milano G, Levi F, et al. Circadian rhythm-varying plasma concentration of 5-fluorouracil during a five-day continuous venous infusion at a constant rate in cancer patients. Cancer Res 1988;48:1676–1680.

208. Fleming RF, Milano G, Thyss A, et al. Correlation between dihydropyrimidine dehydrogenase activity in peripheral mononuclear cells and systemic clearance of fluorouracil in cancer patients. Cancer Res 1982;52:2899–2902.

209. Erlichman C, Fine S, Elhakim T. Plasma pharmacokinetics of 5-FU given by continuous infusion with allopurinol. Cancer Treat Rep 1986;70:903–904.

210. Remick SC, Grem JL, Fischer PH, et al. Phase I trial of 5-fluorouracil and dipyridamole administered by 72-hour concurrent continuous infusion. Cancer Res 1990;50:2667–2672.

211. Grem JL, McAtee N, Steinberg SM, et al. A phase I study of continuous infusion 5-fluorouracil plus calcium leucovorin in combination with n-(phosphonacetyl)-L-aspartate in metastatic gastrointestinal adenocarcinoma. Cancer Res 1993;53:4828–4836.

212. Ensminger WD, Rosowsky A, Raso VO, et al. A clinical pharmacological evaluation of hepatic arterial infusion of 5- fluoro-2′-deoxyuridine and 5-fluorouracil. Cancer Res 1978; 38: 3784–3792.

213. Collins JM, Dedrick RL, King FG, et al. Nonlinear pharmacokinetic models for 5-fluorouracil in man: intravenous and intraperitoneal routes. Clin Pharmacol Ther 1980;28:235–246.

214. Wagner JG, Gyves JW, Stetson PL, et al. Steady-state nonlinear pharmacokinetics of 5-fluorouracil during hepatic arterial and intravenous infusions in cancer patients. Cancer Res 1986;46:1499–1506.

215. Thyss A, Milano G, Renee N, et al. Clinical pharmacokinetic study of 5-FU in continuous 5-day infusions for head and neck cancer. Cancer Chemother Pharmacol 1986;16:64–66.

216. Trump DL, Egorin MJ, Forrest A, et al. Pharmacokinetic and pharmacodynamic analysis of fluorouracil during 72- hour continuous infusion with and without dipyridamole. J Clin Oncol 1991;9:2027–2035.

217. Fety R, Rolland F, Barberi-Heyob M. Clinical impact of pharmacokinetically-guided dose adaptation of 5-fluorouracil: results from a multicentric randomized trial in patients with locally advanced head and neck carcinomas. Clin Cancer Res 1998; 4:2039–2045.

218. Santini J, Milano G, Thyss A, et al. 5-FU therapeutic monitoring with dose adjustment leads to an improved therapeutic index in head and neck cancer. Br J Cancer 1989;59:287–290.

219. Creaven PJ, Rustum YM, Petrelli NJ, et al. Phase I and pharmacokinetic evaluation of floxuridine/leucovorin given on the Roswell Park weekly regimen. Cancer Chemother Pharmacol 1994;34:261–265.

220. Goldberg JA, Kerr DJ, Watson DG, et al. The pharmacokinetics of 5-fluorouracil administered by arterial infusion in advanced colorectal hepatic metastases. Br J Cancer 1990;61:913–915.

221. Sigurdson ER, Ridge JA, Kemeny N. Tumor and liver drug uptake following hepatic artery and portal vein infusion. J Clin Oncol 1987;5:1836–1840.

222. Speyer JL, Collins JM, Dedrick RL, et al. Phase I and pharmacologic studies of 5-fluorouracil administered intraperitoneally. Cancer Res 1980;40:567–572.

223. Sugarbaker PH, Gianola FJ, Speyer JC, et al. Prospective, randomized trial of intravenous versus intraperitoneal 5-fluorouracil in patients with advanced primary colon or rectal cancer. Surgery 1985;98:414–422.

224. Schilsky RL, Choi KE, Grayhack J, et al. Phase I clinical and pharmacologic study of intraperitoneal cisplatin and fluorouracil in patients with advanced intra-abdominal cancer. J Clin Oncol 1990;8:2054–2061.

225. Muggia FM, Chan KK, Russell C, et al. Phase I and pharmacologic evaluation of intraperitoneal 5-fluoro-2′-deoxyuridine. Cancer Chemother Pharmacol 1991;28:241–250.

226. Grem JL, Harold N, Shapiro J, et al. A phase I and pharmacokinetic trial of weekly oral 5-fluorouracil given with eniluracil and low-dose leucovorin. J Clin Oncol 2000;18:3952–3963.

227. Shiotani T, Weber T. Purification and properties of dihydrothymine dehydrogenase from rat liver. J Biol Chem 1981; 256:219–224.

228. Podschun B, Cook PF, Schnackerz KD. Kinetic mechanism of dihydropyrimidine dehydrogenase in pig liver. J Biol Chem 1990;265:12966–12972.

229. Lu Z, Zhang R, Diasio RB. Purification and characterization of dihydropyrimidine dehydrogenase from human liver. J Biol Chem 1992;267:17102–17109.

230. Lu Z, Zhang R, Diasio RB. Comparison of dihydropyrimidine dehydrogenase from human, rat, pig, and cow liver: biochemical and immunological properties. Biochem Pharmacol 1993;46:945–952.

231. Naguib FN, El Kouni MH, Cha S. Enzymes of uracil catabolism in normal and neoplastic tissues. Cancer Res 1985;45: 5405–5412.

232. Ho DH, Townsend L, Luna MA, et al. Distribution and inhibition of dihydrouracil dehydrogenase activities in human tissues using 5-fluorouracil as a substrate. Anticancer Res 1986;6:781–784.

233. Ansfield FJ, Schroeder JM, Curreri AR. Five years clinical experience with 5-fluorouracil. JAMA 1962;181:295–299.

234. Ansfield FJ, Ramirez G, Davis HL, et al. Further clinical studies with intrahepatic arterial infusion with 5-fluorouracil. Cancer 1975;36:2413–2417.

235. Sweeny DJ, Barnes S, Heggie GD, et al. Metabolism of 5-fluorouracil to an n-cholyl-2-fluoro-β-alanine conjugate: previously unrecognized role for bile acids in drug conjugation. Proc Natl Acad Sci U S A 1987;84:5439–5443.

236. Zhang R, Soong S-J, Liu T, et al. Pharmacokinetics and tissue distribution of 2-fluoro-7β-alanine in rats: potential relevance to toxicity pattern of 5-fluorouracil. Drug Metab Dispos 1992; 20:113–119.

237. Dobritzsch D, Schneider G, Schnackerz KD, et al. Crystal structure of dihydropyrimidine dehydrogenase, a major determinant of the pharmacokinetics of the anti-cancer drug 5-fluorouracil. Embo J 2001;20(4):650–660.

238. Etienne MC, Lagrange JL, Dassonville O, et al. Population study of dihydropyrimidine dehydrogenase in cancer patients. J Clin Oncol 1994;12:2248–2253.

239. Lu A, Zhang R, Diasio RB. Dihydropyrimidine dehydrogenase activity in human peripheral blood mononuclear cells and liver: population characteristics, newly identified deficient patients, and clinical implication in 5-fluorouracil chemotherapy. Cancer Res 1993;53:5433–5438.

240. Milano G, Etienne MC, Pierrefite V, et al. Dihydropyrimidine dehydrogenase deficiency and fluorouracil-related toxicity. Br J Cancer 1999;79:627–630.

241. Lu Z, Zhang R, Diasio RB. Population characteristics of hepatic dihydropyrimidine dehydrogenase activity, a key metabolic enzyme in 5-fluorouracil chemotherapy. Clin Pharmacol Ther 1995;58:512–522.

242. Lu Z, Zhang R, Carpenter JT, et al. Decreased dihydropyrimidine dehydrogenase activity in a population of patients with breast cancer: implication for 5-fluorouracil-based chemotherapy. Clin Cancer Res 1998;4:325–329.

243. McLeod HL, Sludden J, Murray GI, et al. Characterization of dihydropyrimidine dehydrogenase in human colorectal tumours. Br J Cancer 1998;77:461–465.

244. McMurrough J, McLeod HL. Analysis of the dihydropyrimidine dehydrogenase polymorphism in a British population. Br J Clin Pharmacol 1996;41:425–427.

245. Chazal M, Etienne MC, Renee N, et al. Link between dihydropyrimidine dehydrogenase activity in peripheral blood mononuclear cells and liver. Clin Cancer Res 1996;2:507–510.

246. Yokota H, Fernandez-Salguero P, Furuya H, et al. cDNA cloning and chromosome mapping of human dihydropyrimidine dehydrogenase, an enzyme associated with 5-fluorouracil toxicity and congenital thymine uraciluria. J Biol Chem 1994;269: 23192–23196.

247. Takai S, Fernandez-Salguero P, Kimura S, et al. Assignment of the human dihydropyrimidine dehydrogenase gene (DPYD) to chromosome region 1p22 by fluorescence in situ hybridization. Genomics 1994;24:613–614.

248. Johnson MR, Diasio RB, Albin N, et al. Structural organization of the human dihydropyrimidine dehydrogenase gene. Cancer Res 1997;57:1660–1663.

249. Ridge SA, Sludden J, Wei X, et al. Dihydropyrimidine dehydrogenase pharmacogenetics in patients with colorectal cancer. Br J Cancer 1998;77:497–500.

250. Fernandez-Salguero PM, Gonzalez FJ, Idle JR, et al. Lack of correlation between phenotype and genotype for the polymorphically expressed dihydropyrimidine dehydrogenase in a family of Pakistani origin. Pharmacogenetics 1997;7: 161–163.

251. Mattison LK, Soong R, Diasio RB. Implications of dihydropyrimidine dehydrogenase on 5-fluorouracil pharmacogenetics and pharmacogenomics. Pharmacogenomics 2002;3:485–492

252. Van Kuilenburg AB, De Abreu RA, van Gennip AH. Pharmacogenetic and clinical aspects of dihydropyrimidine dehydrogenase deficiency. Ann Clin Biochim 2003:40:41–45.

253. van Kuilenburg AB, Vreken P, Abeling NG, et al: Genotype and phenotype in patients with dihydropyrimidine dehydrogenase deficiency. Hum Genet 1999;104:1–9

254. Kirkwood JM, Ensminger W, Rosowsky A, et al. Comparison of pharmacokinetics of 5-fluorouracil and 5-fluorouracil with concurrent thymidine infusions in a phase I trial. Cancer Res 1980;40:107–113.

255. Woodcock TM, Martin DS, Damin LEM, et al. Clinical trials with thymidine and fluorouracil: a phase I and clinical pharmacologic evaluation. Cancer 1980;45:1135–1143.

256. Au JL-S, Rustum YM, Ledesma EJ, et al. Clinical pharmacological studies of concurrent infusion of 5-fluorouracil and thymidine in treatment of colorectal carcinomas. Cancer Res 1982;42:2930–2937.

257. Tuchman M, O'Dea RF, Ramnaraine MLR, et al. Pyrimidine base degradation in cultured murine C-130 neuroblastoma cells and in situ tumors. J Clin Invest 1988;81:425–430.

258. Harvey VJ, Slevin ML, Dilloway MR, et al. The influence of cimetidine on the pharmacokinetics of 5-fluorouracil. Br J Clin Pharmacol 1984;18:421–430.

259. Dilloway MR, Lant AF. Effect of H -receptor antagonists on the pharmacokinetics of 5-fluorouracil in the rat and monkey. Biopharm Drug Dispos 1991;12:17–28.

260. Okuda H, Watabe T, Kawaguchi Y, et al. Lethal drug interactions of sorivudine, a new antiviral drug, with oral 5-fluorouracil prodrugs. Drug Metab Dispos 1997;25:270–273.

261. Yan J, Tyring SK, McCrary MM, et al. The effect of sorivudine on dihydropyrimidine dehydrogenase activity in patients with acute herpes zoster. Clin Pharmacol Ther 1997;61:563–573.

262. Diasio RB. Sorivudine and 5-fluorouracil; a clinically significant drug-drug interaction due to inhibition of dihydropyrimidine dehydrogenase. Br J Clin Pharmacol 1998;46:1–4.

263. Curreri AR, Ansfield FJ, McIvor FA, et al. Clinical studies with 5-fluorouracil. Cancer Res 1958;18:478–484.

264. Ansfield R, Klotz J, Nealon T, et al. A phase III study comparing the clinical utility of four regimens of 5-fluorouracil. Cancer 1977;39:34–40.

265. Poon MA, O'Connell MJ, Moertel CG, et al. Biochemical modulation of fluorouracil: evidence of significant improvement of survival and quality of life in patients with advanced colorectal carcinoma. J Clin Oncol 1989;7:1407–1418.

266. Petrelli N, Douglass HD, Herrera L, et al. The modulation of fluorouracil with leucovorin in metastatic colorectal carcinoma: a prospective randomized phase III trial. J Clin Oncol 1991;7:1419–1426.

267. Seifert P, Baker L, Reed ML, et al. Comparison of continuously infused 5-fluorouracil with bolus injection in treatment of patients with colorectal adenocarcinoma. Cancer 1975;36: 123–128.

268. Rougier P, Paillot B, LaPlanche A, et al. 5-Fluorouracil (5-FU) continuous intravenous infusion compared with bolus administration. Final results of a randomised trial in metastatic colorectal cancer. Eur J Cancer 1997;33:1789–1793

269. Sullivan RD, Young CW, Miller E, et al. The clinical effects of the continuous administration of fluorinated pyrimidines (5-fluorouracil and 5-fluoro-2′-deoxyuridine). Cancer Chemother Rep 1960;8:77–83.

270. Leichman CG, Fleming TR, Muggia FM, et al. Phase II study of fluorouracil and its modulation in advanced colorectal cancer: a Southwest Oncology Group study. J Clin Oncol 1995;13: 1303–1311.

271. Kohne CH, Schoffski P, Wilke H, et al. Effective biomodulation by leucovorin of high-dose infusion fluorouracil given as a weekly 24-hour infusion: results of a randomized trial in patients with advanced colorectal cancer. J Clin Oncol 1998; 16:418–426.

272. O'Dwyer PJ, Manola J Valone FH, et al. Fluorouracil modulation in colorectal cancer: lack of improvement with N -phosphonoacetyl- l -aspartic acid or oral leucovorin or interferon, but enhanced therapeutic index with weekly 24-hour infusion schedule—an Eastern Cooperative Oncology Group/Cancer and Leukemia Group B Study. J Clin Oncol 2001;19:2413–2421.

273. Aranda E, Diaz-Rubio E, Cervantes A, et al. Randomized trial comparing monthly low-dose leucovorin and fluorouracil bolus with weekly high-dose 48-hour continuous-infusion fluorouracil for advanced colorectal cancer: a Spanish Cooperative Group for Gastrointestinal Tumor Therapy (TTD) study. Ann Oncol 1998;9:727–731.

274. Lokich JJ, Ahlgren JD, Gullo JJ, et al. A prospective randomized comparison of continuous infusion fluorouracil with a conventional bolus schedule in metastatic colorectal carcinoma: a Mid-Atlantic Oncology Program Study. J Clin Oncol 1989;7:425–432.

275. de Gramont A, Bosset JF, Milan C, et al. Randomized trial comparing monthly low-dose leucovorin and fluorouracil bolus with bimonthly high-dose leucovorin and fluorouracil bolus plus continuous infusion for advanced colorectal cancer: a French intergroup study. J Clin Oncol 1997;15:808–815.

276. Beerblock K, Rinaldi Y, Andre T, et al. Bimonthly high dose leucovorin and 5-fluorouracil 48-hour continuous infusion in patients with advanced colorectal carcinoma. Groupe d'Etude et de Recherche sur les Cancers de l'Ovaire et Digestifs (GERCOD). Cancer 1997;79:1100–1105.

277. Ansfield F, Ramirez G, Skibba JL, et al. Intrahepatic arterial infusion with 5-fluorouracil. Cancer 1971;28:1147–1151.

278. Kerr DJ, Ledermann JA, McArdle CS, et al. Phase I clinical and pharmacokinetic study of leucovorin and infusional hepatic arterial fluorouracil. J Clin Oncol 1995;13:2968–2972.

279. Sullivan RD, Miller E. The clinical effects of prolonged intravenous infusion of 5-fluoro-2′-deoxyuridine. Cancer Res 1965; 25:1025–1030.

280. Kemeny N, Daly J, Reichman B, et al. Intrahepatic or systemic infusion of fluorodeoxyuridine in patients with liver metastases from colorectal carcinoma. Ann Intern Med 1987;107:459–465.

281. Hohn D, Stagg R, Friedman M, et al. A randomized trial of continuous intravenous versus hepatic intraarterial floxuridine in patients with colorectal cancer metastatic to the liver: the Northern California Oncology Group trial. J Clin Oncol 1989;7:1646–1654.

282. Anderson N, Lokich J, Bern M, et al. A phase I clinical trial of combined fluoropyrimidines with leucovorin in a 14-day infusion. Demonstration of biochemical modulation. Cancer 1989;63:233–237.

283. Martin JK, O'Connell MJ, Wieand HS, et al. Intra-arterial floxuridine vs systemic fluorouracil for hepatic metastases from colorectal cancer. Arch Surg 1990;125:1022–1027.

284. Creaven PJ, Rustum YM, Petrelli NJ, et al. Phase I and pharmacokinetic evaluation of floxuridine/leucovorin given on the Roswell Park weekly regimen. Cancer Chemother Pharmacol 1994;34:261–265

285. Vokes EE, Raschko JW, Vogelzang NJ, et al. Five day infusion of fluorodeoxyuridine with high-dose oral leucovorin: a phase I study. Cancer Chemother Pharmacol 1991;28:69–73.

286. Brennan MJ, Waitkevicius VK, Rebuck JW. Megaloblastic anemia associated with inhibition of thymine synthesis (observations during 5-fluorouracil treatment). Blood 1960;14:1535–1545.

287. Kelvin FM, Gramm HF, Gluck WL, et al. Radiologic manifestations of small-bowel toxicity due to floxuridine therapy. AJR Am J Roentgenol 1986;146:39–43.

288. Benson AB III, Ajana JA, Catalano RB, et al. Recommended guidelines for the treatment of cancer treatment-induced diarrhea. J Clin Oncol 2004;22:2918–2926.

289. Loprinzi CL, Cianflone SG, Dose AM, et al. A controlled evaluation of an allopurinol mouthwash as prophylaxis against 5-fluorouracil induced stomatis. Cancer 1990;65:1879–1882.

290. Mahood DJ, Kose AM, Loprinzi CL, et al. Inhibition of fluorouracil-induced stomatitis by oral cryotherapy. J Clin Oncol 1991;9:449–452.

291. DeSpain JD. Dermatologic toxicity of chemotherapy. Semin Oncol 1992;19:501–507.

292. Vukelja SJ, Bonner MW, McCollough M, et al. Unusual serpentine hyperpigmentation associated with 5-fluorouracil. Case report and review of cutaneous manifestations associated with systemic 5-fluorouracil. J Am Acad Dermatol 1991;25:905–908.

293. Pujol RM, Rocamora V, Lopez-Pousa A, et al. Persistent supravenous erythematous eruption. A rare local complication of intravenous 5-fluorouracil therapy. J Am Acad Dermatol 1998;39:839–842.

294. Riehl JL, Brown WJ. Acute cerebellar syndrome secondary to 5-fluorouracil therapy. Neurology 1964;14:961–967.

295. Moertel CG, Reitemeier RJ, Bolton CF, et al. Cerebellar ataxia associated with fluorinated pyrimidine therapy. Cancer Chemother Rep 1964;41:15–18.

296. Lynch HT, Droszcz CP, Albano WA, et al. “Organic brain syndrome” secondary to 5-fluorouracil toxicity. Dis Colon Rectum 1981;24:130–131.

297. Moore DH, Fowler WC Jr, Crumpler LS. 5-Fluorouracil neurotoxicity. Gynecol Oncol 1990;36:152–154.

298. Tuxen MK, Hansen SW. Neurotoxicity secondary to antineoplastic drugs. Cancer Treat Rev 1994;20:191–214.

299. Bygrave HA, Geh JI, Jani Y, et al. Neurological complications of 5-fluorouracil chemotherapy. Case report and review of the literature. Clin Oncol 1998;10:334–336.

300. O'Connell MJ, Powis G, Rubin J, et al. Pilot study of PALA and 5-FU in patients with advanced cancer. Cancer Treat Rep 1982;66:77–80.

301. Wooley PV, Ayoob MJ, Smith FP, et al. A controlled trial of the effect of 4-hydroxypyrazolopyrimidine (Allopurinol) on the toxicity of a single bolus dose of 5-fluorouracil. J Clin Oncol 1985;3:103–109.

302. Muggia FM, Camacho FJ, Kaplan BH, et al. Weekly 5-fluorouracil combined with PALA. Toxic and therapeutic effects in colorectal cancer. Cancer Treat Rep 1987;71:253–256.

303. Diasio RB, Beavers TL, Carpenter T. Familial deficiency of dihydropyrimidine dehydrogenase: biochemical basis for familial pyrimidinemia and severe 5-fluorouracil-induced toxicity. J Clin Invest 1998;81:47–51.

304. Harris BE, Carpenter JT, Diasio RB. Severe 5-fluorouracil toxicity secondary to dihydropyrimidine dehydrogenase deficiency. Cancer Res 68:499–501, 1991

305. Takimoto CH, Lu Z-H, Zhang R, et al. Severe neurotoxicity following 5-fluorouracil-based chemotherapy in a patient with dihydropyrimidine dehydrogenase deficiency. Clin Cancer Res 1996;2:477–481.

306. Hook CC, Kimmel DW, Kvols LK, et al. Multifocal inflammatory leukoencephalopathy with 5-fluorouracil and levamisole. Ann Neurol 1992;31:262–267.

307. Figueredo AT, Fawcet SE, Molloy DW, et al. Disabling encephalopathy during 5-fluorouracil and levamisole adjuvant therapy for resected colorectal cancer. A report of two cases. Cancer Invest 1995;13:608–611.

307a. Luppi G, Zoboli A, Barbieri F, et al. Multifocal leukoencephalopathy associated with 5-fluorouracil and levamisole adjuvant therapy for colon cancer. A report of two cases and review of the literature. The INTACC Intergruppo Nazionale Terpia Adiuvante Colon Carcinoma. Ann Oncol 1996;7:412–415.

308. Neuwelt EA, Barnet PA, Glasberg M, et al. Neurotoxicity of chemotherapeutic agents after blood-brain barrier modification neuropathological studies. Ann Neurol 1983;14:316–324.

309. Davis ST, Joyner SS, Baccanari DP, et al. 5-Ethynyluracil (776C85). Protection from 5-fluorouracil-induced neurotoxicity in dogs. Biochem Pharmacol 1994;48:233–236.

310. Okada R, Shibutani M, Matsuo T, et al. Experimental neurotoxicity of 5-fluorouracil and its derivatives is due to poisoning by the monofluorinated organic metabolites, monofluoroacetic acid and “-fluoro-β-alanine. Acta Neuropathol 1990; 81:66–73.

311. Berg SL, Balis FM, McCully CL, et al. Intrathecal 5-fluorouracil in the rhesus monkey. Cancer Chemother Pharmacol 1992;31: 127–130.

312. Yamada M, Nakagawa H, Fukushima M, et al. In vitro study on intrathecal use of 5-fluoro-2′-deoxyuridine (FdUrd) for meningeal dissemination of malignant brain tumors. J Neurooncol 1998;37:115–121.

313. Tsavaris N, Kosmas C, Vadiaka M, et al. Cardiotoxicity following different doses and schedules of 5fluorouracil administration for malignancy—a survey of 427 pattients. Med Sci Monit 2002;8:151–157.

314. Becker K, Erckenbrecht JF, Haussinger D, et al. Cardiotoxicity of the antiproliferative compound fluorouracil. Drugs 1999;57: 475–484.

315. Meyer CC, Calis KA, Burke LB, et al. Symptomatic cardiotoxicity associated with 5-fluorouracil. Pharmacotherapy 1997;17: 729–736.

316. Grandi AM, Pinotti G, Morandi E, et al. Noninvasive evaluation of cardiotoxicity of 5-fluorouracil and low doses of folinic acid. A one-year follow-up study. Ann Oncol 1997;8:705–708.

317. Wang WS, Hsieh RK, Chiou TJ, et al. Toxic cardiogenic shock in a patient receiving weekly 24-hr infusion of high-dose 5-fluorouracil and leucovorin. Jpn J Clin Oncol 1998;28:551–554.

318. Arrellano M, Malet-Martino M, Martine R, et al. The anti-cancer drug 5-fluorouracil is metabolized by the isolated perfused rat liver and in rats into highly toxic fluoroacetate. Br J Cancer 1998;77:79–86.

318a. Lemaire L, Malet-Martino MC, de Forni M, et al. Cardiotoxicity of commercial 5-fluorouracil stems from the alkaline hydrolysis of this drug. Br J cancer 1992;66:119–127.

319. Porta C, Moroni M, Ferrari S, et al. Endothelin-1 and 5-fluorouracil-induced cardiotoxicity. Neoplasma 1998;45:81–82.

320. Mosseri M, Fingert HJ, Varticovoski L, et al. In vitro evidence that myocardial ischemia resulting from 5-fluorouracil chemotherapy is due to protein kinase C-mediated vasoconstriction of vascular smooth muscle. Cancer Res 1993;53:3028–3033.

321. al-Tweigeri T, Nabholtz JM, Mackey JR. Ocular toxicity and cancer chemotherapy. A review. Cancer 1996;78:1359–1373.

322. Eiseman AS, Flanagan JC, Brooks AB, et al. Ocular surface, ocular adnexal, and lacrimal complications associated with the use of systemic 5-fluorouracil. Ophthal Plast Reconstr Surg 2003;19:216–224.

323. Loprinzi CL, Wender DB, Veeder MH, et al. Inhibition of 5-fluorouracil-induced ocular irritation by ocular ice packs. Cancer 1994;74:945–948.

324. Wong MK, Bjarnason GA, Hrushesky WJ, et al. Steroid- responsive interstitial lung disease in patients receiving 2′- deoxy-5-fluorouridine infusion chemotherapy. Cancer 1995;75:2558–2564.

325. Boyle FM, Smith RC, Levi JA. Continuous hepatic artery infusion of 5-fluorouracil for metastatic colorectal cancer localised to the liver. Aust N Z J Med 1993;23:32–34.

326. Hohn DC, Rayner AA, Economou JS, et al. Toxicities and complications of implanted pump hepatic arterial and intravenous floxuridine infusion. Cancer 1986;57:465–470.

327. Kemeny N, Seiter K, Niedzwiecki D, et al. A randomized trial of intrahepatic infusion of fluorodeoxyuridine with dexamethasone versus fluorodeoxyuridine alone in the treatment of metastatic colorectal cancer. Cancer 1992;69:327–334.

328. Kemeny N, Seiter K, Conti JA, et al. Hepatic arterial floxuridine and leucovorin for unresectable liver metastases from colorectal carcinoma. New dose schedules and survival update. Cancer 1994;73:1134–1142.

329. Kemeny N, Conti JA, Cohen A, et al. Phase II study of hepatic arterial floxuridine, leucovorin, and dexamethasone for unresectable liver metastases from colorectal carcinoma. J Clin Oncol 1994;12:2288–2295.

330. Stein BN, Petrelli NJ, Douglass HO, et al. Age and sex are independent predictors of 5-fluorouracil toxicity. Cancer 1995; 75:11–17.

331. Zalcberg J, Kerr D, Seymour L, et al. Haematological and non-haematological toxicity after 5-fluorouracil and leucovorin in patients with advanced colorectal cancer is significantly associated with gender, increasing age and cycle number. Tomudex International Study Group. Eur J Cancer 1998;34:1871–1875.

332. Tepper JE, O'Connell MJ, Petroni GR, et al. Adjuvant post-operative fluorouracil-modulated chemotherapy combined with pelvic radiation therapy for rectal cancer. Initial results of intergroup 0114. J Clin Oncol 1997;15:2030–2039.

333. Toxicity of fluorouracil in patients with advanced colorectal cancer. Effect of administration schedule and prognostic factors. Meta-analysis group in cancer. J Clin Oncol 1988;16:3537–3541.

334. Sloan JA, Goldberg RM, Sargent DJ, et al. Women experience greater toxicity with fluorouracil-based chemotherapy for colorectal cancer J Clin Oncol 2002;20:1491–1498.

335. Tsalic M, Bar-Sela G, Beny A, et al. Severe toxicity related to the 5-fluorouracil/leucovorin combination (the Mayo Clinic regimen): a prospective study in colorectal cancer patients. Am J Clin Oncol 2003;26:103–106.

336. Port RE, Daniel B, Ding RW, et al. Relative importance of dose, body surface area, sex and age for 5-fluorouracil clearance. Oncology 1991;48:277–281.

337. Milano G, Etienne MC, Cassuto-Viguier E, et al. Influence of sex and age on fluorouracil clearance. J Clin Oncol 1992;10: 1171–1175.

338. Etienne MC, Chatelut E, Pivot X, et al. Co-variables influencing 5-fluorouracil clearance during continuous venous infusion. A NONMEM analysis. Eur J Cancer 1998;34:92–97

339. Wildiers H, Highley MS, de Bruijn EA, Van Oosterom AT. Pharmacology of anticancer drugs in the elderly population. Clin Pharmacokinet 2003;42:1213–1242

340. Modulation of fluorouracil by leucovorin in patients with advanced colorectal cancer. Evidence in terms of response rate. The advanced colorectal cancer meta-analysis project. J Clin Oncol 1992;10:896–903.

341. Meta-analysis of randomized trials testing the biochemical modulation of fluorouracil by methotrexate in metastatic colorectal cancer. The advanced colorectal cancer meta-analysis project. J Clin Oncol 1994;12:960–969.

342. Efficacy of intravenous continuous infusion of fluorouracil compared with bolus administration in advanced colorectal cancer. Meta-analysis group in cancer. J Clin Oncol 1998;16:301–308.

343. Reappraisal of hepatic arterial infusion in the treatment of nonresectable liver metastases from colorectal cancer. Meta- analysis group in cancer. J Natl Cancer Inst 1996;88:252–258.

344. Thirion P, Piedbois P, Buyse M, et al. Alpha-interferon does not increase the efficacy of 5-fluorouracil in advanced colorectal cancer. Br J Cancer 2001;84:611–620.

345. Fox RM, Woods RL, Tattersall MHN. Allopurinol modulation of high-dose fluorouracil toxicity. Cancer Treat Rev 1979;6 (Suppl):143–147.

346. Campbell TN, Howell SB, Pfeifle C, et al. High-dose allopurinol modulation of 5-FU toxicity. Phase I trial of an outpatient dose schedule. Cancer Treat Rep 1982;66:1723–1727.

347. Howell SB, Pfeifle CE, Wung WE. Effect of allopurinol on the toxicity of high-dose 5-fluorouracil administered by intermittent bolus injection. Cancer 1983;51:220–225.

348. Yoshida M, Hoshi A, Kuretani K. Prevention of antitumor effect of 5-fluorouracil by hypoxanthine. Biochem Pharmacol 1978; 27:2979–2982.

349. Yoshida M, Hoshi A. Mechanism of inhibition of phosphoribosylation of 5-fluorouracil by purines. Biochem Pharmacol 1984;33:2863–2867.

350. Martin DS, Stolfi RL, Sawyer RC, et al. High-dose 5-fluorouracil with delayed uridine “rescue” in mice. Cancer Res 1982;42: 3864–3970.

351. Sawyer RC, Stolfi RL, Spiegelman S, et al. Effect of uridine on the metabolism of 5-fluorouracil in the CD8F1 murine mammary carcinoma system. Pharm Res 1984;2:69–75.

352. Klubes P, Cerna I. Use of uridine rescue to enhance the antitumor selectivity of 5-fluorouracil. Cancer Res 1983;43: 3182–3186.

353. Peters GJ, van Dijk J, Laurensse E, et al. In vitro biochemical and in vivo biological studies of the uridine “rescue” of 5-fluorouracil. Br J Cancer 1988;57:259–265.

354. Nord LK, Stolfi RL, Martin DS. Biochemical modulation of 5-fluorouracil with leucovorin or delayed uridine rescue. Biochem Pharmacol 1992;43:2543–2549.

355. Leyva A, van Groeningen CJ, Kraal I, et al. Phase I and pharmacokinetic studies of high-dose uridine intended for rescue from 5-fluorouracil toxicity. Cancer Res 1984;44:5928–5933.

356. van Groeningen CJ, Leyva A, Kraal I, et al. Clinical and pharmacokinetic studies of prolonged administration of high-dose uridine intended for rescue from 5-FU toxicity. Cancer Treat Rep 1986;70:745–750.

357. van Groeningen CJ, Peters GJ, Leyva A, et al. Reversal of 5-fluorouracil-induced myelosuppression by prolonged administration of high-dose uridine. J Natl Cancer Inst 1989;81: 157–162.

358. Martin DS, Stolfi RL, Sawyer RC. Utility of oral uridine to substitute for parenteral uridine rescue of 5-fluorouracil therapy, with and without a uridine phosphorylase inhibitor (5-benzylacyclouridine). Cancer Chemother Pharmacol 1989;24:9–14.

359. Klubes P, Geffen DB, Cysyk RL. Comparison of the bioavailability of uridine in mice after either oral or parenteral administration. Cancer Chemother Pharmacol 1986;17:236–240.

360. van Groeningen CJ, Peters GJ, Nadal JC, et al. Clinical and pharmacological study of orally administered uridine. J Natl Cancer Inst 1991;83:437–441.

361. Burns ER, Beland SS. Effect of biological time on the determination of the LD50 of 5-fluorouracil in mice. Pharmacology 1984;28:296–300.

362. Peters GJ, van Dijk J, Nadal JC, et al. Diurnal variation in the therapeutic efficacy of 5-fluorouracil against murine colon cancer. In Vivo 187;1:113–118.

363. van Roemeling R, Hrushesky WJM. Determination of the therapeutic index of floxuridine by its circadian infusion pattern. J Natl Cancer Inst 1990;82:386–393.

364. Minshull M, Gardner MLG. The effects of time of administration of 5-fluorouracil on leucopenia in the rat. Eur J Cancer Clin Oncol 1984;20:857–858.

365. Zhang R, Lu Z, Liu T, et al. Relationship between circadian-dependent toxicity of 5-fluorodeoxyuridine and circadian rhythms of pyrimidine enzymes. Possible relevance to fluoropyrimidine therapy. Cancer Res 1993;53:2816–2822.

366. Burns ER. Circadian rhythmicity in DNA synthesis in untreated and saline-treated mice as a basis for improved chemotherapy. Cancer Res 1981;41:2795–2802.

367. Burns ER, Beland SS. Induction by 5-fluorouracil of a major phase difference in the circadian profiles of DNA synthesis between the Ehrlich ascites carcinoma and five normal organs. Cancer Lett 1983;20:235–239.

368. Harris BE, Song R, He YJ, et al. Circadian rhythm of rat liver dihydropyrimidine dehydrogenase. Biochem Pharmacol 1988; 37:4759–4762.

369. Zhang R, Lu Z, Liu T, et al. Circadian rhythm of rat spleen cytoplasmic thymidine kinase. Biochem Pharmacol 1993;45: 1115–1119.

370. Smaaland R, Laerum OD, Lote K, et al. DNA synthesis in human bone marrow is circadian stage dependent. Blood 1991;77:2603–2611.

371. Buchi KN, Moore JG, Hrushesky WJM, et al. Circadian rhythm of cellular proliferation in human rectal mucosa. Gastroenterology 1991;101:410–415.

372. Grem JL, Yee LK, Venzon DJ, et al. Inter- and intraindividual variation in dihydropyrimidine dehydrogenase activity in peripheral blood mononuclear cells. Cancer Chemother Pharmacol 1997;40:117–125.

373. Takimoto CH, Yee LK, Venzon D, et al. High inter- and intrapatient variation in 5-fluorouracil plasma concentrations during a prolonged drug infusion. Clin Cancer Res 1999;5:1347–1352.

374. Metzger G, Massari C, Etienne MC, et al. Spontaneous or imposed circadian changes in plasma concentrations of 5- fluorouracil coadministered with folinic acid and oxaliplatin. Relationship with mucosal toxicity in patients with cancer. Clin Pharmacol Ther 1994;56:190–201.

375. von Roemeling R, Hrushesky WJM. Circadian patterning of continuous floxuridine infusion reduces toxicity and allows higher dose intensity in patients with widespread cancer. J Clin Oncol 1989;7:1710–1719.

376. Hrushesky WJM, von Roemeling R, Lanning TM, et al. Circadian-shaped infusions of floxuridine for progressive metastatic renal cell carcinoma. J Clin Oncol 1990;8:1504–1513.

377. Wesen C, Hrushesky WJM, van Roemeling R. Circadian modification of intra-arterial 5-fluoro-2′-deoxyuridine infusion rate reduces its toxicity and permits higher dose-intensity. J Infus Chemother 1992;2:69–75.

378. Levi FA, Zidani R, Vannetzel JM, et al. Chronomodulated versus fixed-infusion-rate delivery of ambulatory chemotherapy with oxaliplatin, fluorouracil, and folinic acid (leucovorin) in patients with colorectal cancer metastases. A randomized multi-institutional trial. J Natl Cancer Inst 1994;86:1608–1617.

379. Levi F, Zidani R, Misset JL. Randomised multicentre trial of chronotherapy with oxaliplatin, fluorouracil, and folinic acid in metastatic colorectal cancer. International organization for cancer chronotherapy. Lancet 1997;350:681–686.

380. Giacchetti S, Bjarnason G, Garufi C, et al. First line infusion of 5-fluorouracil, leucovorin and oxaliplatin for metastatic colorectal cancer: 4-day chronomodulated (FFL4-10) versus 2-day FOLFOX2. A multicenter randomized Phase III trial of the Chronotherapy Group of the European Organization for Research and Treatment of Cancer (EORTC 05963). Proc Am Soc Clin Oncol 2004;22(14Suppl): abstr 3526.

381. Bjarnason GA, Kerr IG, Doyle N, et al. Phase I study of 5-fluorouracil by a 14-day circadian infusion in metastatic adenocarcinoma patients. Cancer Chemother Pharmacol 1993; 33:221–228.

382. Bertino JR. Biomodulation of 5-fluorouracil with antifolates. Semin Oncol 1997;24(Suppl 18):52–56.

383. Benz C, Tillis T, Tattelman E, et al. Optimal scheduling of methotrexate and 5-fluorouracil in human breast cancer. Cancer Res 1982;42:2081–2086.

384. Donehower RC, Allegra JC, Lippman ME, et al. Combined effects of methotrexate and 5-fluoropyrimidines on human breast cancer cells in serum-free tissue culture. Eur J Cancer 1980;16:655–661.

385. Piper AA, Nott SE, Mackinnon WB, et al. Critical modulation by thymidine and hypoxanthine of sequential methotrexate-5-fluorouracil synergism in murine L1210 cells. Cancer Res 1983; 43:5101–5105.

386. Sawyer RC, Stolfi RL, Martin DS, et al. Inhibition by methotrexate of the stable incorporation of 5-fluorouracil into murine bone marrow DNA. Biochem Pharmacol 1989;38:2305–2311.

387. McSheehy PMJ, Prior MJW, Griffiths JR. Enhanced 5-fluorouracil cytotoxicity and elevated 5-fluoronucleotides in the rat walker carcinosarcoma following methotrexate pre-treatment. A 19 F-MRS study in vivo. Br J Cancer 1992;65:369–375.

388. Tattersall MHN, Jackson RC, Connors TA, et al. Combination chemotherapy. The interaction of methotrexate and 5-fluorouracil. Eur J Cancer 1973;9:733–739.

389. Bertino JR, Sawicki WL, Linquist CA, et al. Schedule- dependent antitumor effects of methotrexate and 5-fluorouracil. Cancer Res 1977;37:327–328.

390. Kemeny N, Ahmed T, Michaelson R, et al. Activity of sequential low-dose methotrexate and fluorouracil in advanced colorectal carcinoma. Attempt at correlation with tissue and blood levels of phosphoribosylpyrophosphate. J Clin Oncol 1984;2:311–315.

391. Marsh JC, Bertino JR, Katz KH, et al. The influence of drug interval on the effect of methotrexate and fluorouracil in the treatment of advanced colorectal cancer. J Clin Oncol 1991;9: 371–380.

392. Browman GP, Levine MN, Goodyear MD, et al. Methotrexate/ fluorouracil scheduling influences normal tissue toxicity but not antitumor effects in patients with squamous cell head and neck cancer. Results from a randomized trial. J Clin Oncol 1988;6:963–968.

393. Elliot WL, Howeard CT, Kykes DJ, et al. Sequence and schedule-dependent synergy of trimetrexate in combination with 5-fluorouracil in vitro and in mice. Cancer Res 1989;15:5586–5590.

394. Romanini A, Li WW, Colofiore JR, et al. Leucovorin enhances cytotoxicity of trimetrexate/fluorouracil, but not methotrexate/ fluorouracil, in CCRF-CEM cells. J Natl Cancer Inst 1992; 84:1033–1038.

395. Punt CJ, Blanke CD, Zhang J, et al. Integrated analysis of overall survival in two randomised studies comparing 5-fluorouracil/leucovorin with or without trimetrexate in advanced colorectal cancer. Ann Oncol 2002;13(1):92–94.

396. Danenberg PV, Danenberg KD. Effect of 5,10-methylenetetrahydrofolate and the dissociation of 5-fluorodeoxyuridylate binding of human thymidylate synthetase. Evidence for an ordered mechanism. Biochemistry 1978;17:4018–4024.

397. Houghton JA, Maroda SJ, Phillips JO, et al. Biochemical determinants of responsiveness to 5-fluorouracil and its derivatives in xenografts of human colorectal adenocarcinomas in mice. Cancer Res 1981;41:144–149.

398. Houghton JA, Torrance PM, Radparvar S, et al. Binding of 5-fluorodeoxyuridylate to thymidylate synthase in human colon adenocarcinoma xenografts. Eur J Cancer Clin Oncol 1986;22:505–510.

399. Spears CP, Gustavsson BG, Fiosing R. Folinic acid modulation of fluorouracil. Tissue kinetics of bolus administration. Invest New Drugs 1989;7:27–36.

400. Evans RM, Laskin JD, Hakala MT. Effects of excess folates and deoxyinosine on the activity and site of action of 5-fluorouracil. Cancer Res 1981;41:3288–3295.

401. Yin M-B, Zakrzewski SF, Hakala MT. Relationship of cellular folate cofactor pools to the activity of 5-fluorouracil. Mol Pharmacol 1983;23:190–197.

402. Cao S, Frank C, Rustum YM. Role of fluoropyrimidine Schedule and (6R, S)leucovorin dose in a preclinical animal model of colorectal carcinoma. J Natl Cancer Inst 1996;88:430–436.

403. Drake JC, Voeller DM, Allegra CJ, et al. The effect of dose and interval between 5-fluorouracil and leucovorin on the formation of thymidylate synthase ternary complex in human cancer cells. Br J Cancer 1995;71:1145–1150.

404. Keyomarsi K, Moran R. Folinic acid augmentation of the effects of fluoropyrimidines on murine and human leukemic cells. Cancer Res 1986;46:5229–5235.

405. Matherly LH, Czaijkowski CA, Muench SP, et al. Role for cytosolic folate binding proteins in compartmentation of endogenous tetrahydrofolates and the formyltetrahydrofolate-mediated enhancement of 5-fluoro-2′-deoxyuridine antitumor activity in vitro. Cancer Res 1990;50:3262–3269.

406. Nadal JC, van Groeningen CJ, Pinedo HM, et al. In vivo potentiation of 5-fluorouracil by leucovorin in murine colon carcinoma. Biomed Pharmacother 1988;42:387–393.

407. Radparvar S, Houghton PJ, Houghton JA. Effect of polyglutamylation of 5,10-methylenetetrahydrofolate on the binding of 5-fluoro-2-deoxyuridylate to thymidylate synthase purified from a human colon adenocarcinoma xenograft. Biochem Pharmacol 1989;38:335–342.

408. Wright JE, Dreyfuss A, El-Magharbel I, et al. Selective expansion of 5,10-methylenetetrahydrofolate pools and modulation of 5-fluorouracil antitumor activity by leucovorin in vivo. Cancer Res 1989;49:2592–2596.

409. Carlsson G, Gustavsson BG, Spears CP, et al. 5-Fluorouracil plus leucovorin as adjuvant treatment of an experimental liver tumor in rats. Anticancer Res 1990;10:813–816.

410. Houghton JA, Williams LG, Cheshire PJ, et al. Influence of dose of [6RS]-leucovorin on reduced folate pools and 5-fluorouracil-mediated thymidylate synthase inhibition in human colon adenocarcinoma xenografts. Cancer Res 1990; 50:3940–3946.

411. Houghton JA, Williams WG, deGraaf SS, et al. Comparison of the conversion of 5-formyltetrahydrofolate and 5- methyltetrahydrofolate to 5,10-methylenetetrahydrofolates and tetrahydrofolates in human colon tumors. Cancer Commun 1989;1: 167–174.

412. Bertrand R, Jolivet J. Lack of interference by the unnatural isomer of 5-formyltetrahydrofolate with the effects of the natural isomer in leucovorin preparations. J Natl Cancer Inst 1989;81: 1175–1178.

413. Straw JA, Szapary D, Wynn WT. Pharmacokinetics of the diastereoisomers of leucovorin after intravenous and oral administration to normal subjects. Cancer Res 1984;44:3114–3119.

414. Machover D, Goldschmidt E, Chollet P, et al. Treatment of advanced colorectal and gastric adenocarcinomas with 5-fluorouracil and high-dose folinic acid. J Clin Oncol 1986;4:685–696.

415. Trave F, Rustum YM, Petrelli NJ, et al. Plasma and tumor tissue pharmacology of high dose intravenous leucovorin calcium in combination with fluorouracil in patients with advanced colorectal carcinoma. J Clin Oncol 1988;6:1181–1188.

416. Newman EA, Straw JA, Doroshow JH. Pharmacokinetics of diastereoisomers of (6R, S)-folinic acid (leucovorin) in humans during constant high-dose intravenous infusion. Cancer Res 1989;49:5755–5760.

417. Priest DG, Schmitz JC, Bunni MA, et al. Pharmacokinetics of leucovorin metabolites in human plasma as a function of dose administered orally and intravenously. J Natl Cancer Inst 1991; 83:1806–1812.

418. Haller DG, Catalano PJ, Macdonald JS, et al. Fluorouracil, leucovorin and levamisole adjuvant therapy for colon cancer. Five-year final report of INT-0089. Proc Am Soc Clin Oncol 1998; 17:265a.

419. Elias L, Crissman HA. Interferon effects upon the adenocarcinoma MCA 38 and HL-60 cell lines. Antiproliferative responses and synergistic interactions with halogenated pyrimidine antimetabolites. Cancer Res 1988;48:4868–4873.

420. Elias L, Sandoval JM. Interferon effects upon fluorouracil metabolism by HL-60 cells. Biochem Biophys Res Commun 1989;163:867–874.

421. Schwartz EL, Hoffman M, O'Connor CJ, et al. Stimulation of 5-fluorouracil metabolic activation by interferon-” in human colon carcinoma cells. Biochem Biophys Res Commun 1992; 182:1232–1239.

422. Schwartz EL, Baptiste N, O'Connor CJ, et al. Potentiation of the antitumor activity of 5-fluorouracil in colon carcinoma cells by the combination of interferon and deoxyribonucleosides results from complementary effects on thymidine phosphorylase. Cancer Res 1994;54:1472–1478.

423. Morita T, Tokue A. Biomodulation of 5-fluorouracil by interferon-alpha in human renal carcinoma cells. Relationship to the expression of thymidine phosphorylase. Cancer Chemother Pharmacol 1999;44:91–96.

424. Houghton JA, Adkins DA, Rahman A, et al. Interaction between 5-fluorouracil, [6R, S]leucovorin, and recombinant human interferon-”2a in cultured colon adenocarcinoma cells. Cancer Commun 1991;3:225–231.

425. Houghton JA, Morton CL, Adkins DA, et al. Locus of the interaction among 5-fluorouracil, leucovorin and interferon-”2a in colon carcinoma cells. Cancer Res 1993;53:4243–4250.

426. Neefe JR, Glass J. Abrogation of interferon-induced resistance to interferon-activated major histocompatibility complex-unrestricted killers by treatment of a melanoma cell line with 5-fluorouracil. Cancer Res 1991;51:3159–3163.

427. Reiter Z, Ozes ON, Blatt LM, et al. A dual antitumor effect of a combination of interferon-” or interleukin-2 and 5-fluorouracil on natural killer (NK) cell-mediated cytotoxicity. Clin Immunol Immunopathol 1992;62:103–111.

428. Koshiji M, Adachi Y, Taketani S, et al. Mechanisms underlying apoptosis induced by combination of 5-fluorouracil and interferon-gamma. Biochem Biophys Res Commun 1997; 240: 376–381.

429. van der Wilt CL, Smid K, Aherne GW, et al. Biochemical mechanisms of interferon modulation of 5-fluorouracil activity in colon cancer cells. Eur J Cancer 1997;33:471–478.

430. Ismail A, Van Groeningen CJ, Hardcastle A, et al. Modulation of fluorouracil cytotoxicity by interferon-alpha and -gamma. Mol Pharmacol 1998;53:252–261.

431. Danhauser LL, Freimann JH Jr, Gilchrist TL, et al. Phase I and plasma pharmacokinetic study of infusional 5-fluorouracil combined with recombinant interferon alfa-2a in patients with advanced cancer. J Clin Oncol 1993;11:751–761.

432. Schuller J, Czejka M. Pharmacokinetic interaction of 5-fluorouracil and interferon alpha-2b with or without folinic acid. Med Oncol 1995;12:47–53.

433. Yee LK, Allegra CJ, Steinberg SM, et al. Decreased catabolism of 5-fluorouracil in peripheral blood mononuclear cells during therapy with 5-fluorouracil, leucovorin, and interferon “-2a. J Natl Cancer Inst 1992;84:1820–1825.

434. Grem JL, Quinn M, Ismail AS, et al. Pharmacokinetics and pharmacodynamic effects of 5-fluorouracil given as a one-hour intravenous infusion. Cancer Chemother Pharmacol 2001; 47:117–125.

435. O'Dwyer PJ, King SA, Hoth DF, et al. Role of thymidine in biochemical modulation. A review. Cancer Res 1987;47: 3911–3919.

436. Vogel SJ, Presant CA, Ratkin FA, et al. Phase I study of thymidine plus 5-fluorouracil infusions in advanced colorectal carcinoma. Cancer Treat Rep 1979;63:1–5.

437. Chen J-J, Jones ME. Effect of 6-azauridine on de novo pyrimidine biosynthesis in cultured Ehrlich ascites cells. J Biol Chem 1979;254:4908–4914.

438. Ahluwalia GS, Grem JL, Ho Z, et al. Metabolism and action of amino acid analog anti-cancer agents. Pharmacol Ther 1990; 46:243–271.

439. O'Dwyer PJ, Alonso MT, Leyland-Jones B. Acivicin. A new glutamine antagonist in clinical trials. J Clin Oncol 1984; 2:1064–1071.

440. Pizzorno G, Wiegand RA, Lentz SK, et al. Brequinar potentiates 5-fluorouracil antitumor activity in a murine model colon 38 tumor by tissue-specific modulation of uridine nucleotide pools. Cancer Res. 1992;52:1660–1665.

441. Moyer JD, Smith PA, Levy EJ, et al. Kinetics of n-(phosphonacetyl)-l-aspartate and pyrazofurin depletion of pyrimidine ribonucleotide and deoxyribonucleotide pools and their relationship to nucleic acid synthesis in intact and permeabilized cells. Cancer Res 1982;42:4525–4531.

442. Ardalan B, Glazer RI, Kensler TW, et al. Synergistic effect of 5-fluorouracil and n-(phosphonacetyl)-l-aspartate on cell growth and ribonucleic acid synthesis in human mammary carcinoma. Biochem Pharmacol 1981;30:2045–2049.

443. Liang C-M, Donehower RC, Chabner BA. Biochemical interactions between n-(phosphonacetyl)-L-aspartate and 5- fluorouracil. Mol Pharmacol 1982;21:224–230.

444. Martin DS, Stolfi RL, Sawyer RC, et al. Therapeutic utility of utilizing low doses of n-(phosphonacetyl)-l-aspartic acid in combination with 5-fluorouracil. A murine study with clinical relevance. Cancer Res 1983;43:2317–2321.

445. Grem JL, King SA, O'Dwyer PJ, et al. Biochemistry and clinical activity of n-(phosphonacetyl)-l-aspartate. A review. Cancer Res 1988;48:4441–4454.

446. Casper ES, Vale K, Williams LJ, et al. Phase I and clinical pharmacological evaluation of biochemical modulation of 5-fluorouracil with n-(phosphonacetyl)-l-aspartic acid. Cancer Res 1983;43:2324–2329.

447. Nabeya Y, Isono K, Moriyama Y, et al. Ribose-transfer activity from uridine to 5-fluorouracil in Ehrlich ascites tumor cells. Jpn J Cancer Res 1990;81:692–700.

448. Moran RG, Danenberg PV, Heidelberger C. Therapeutic response of leukemic mice treated with fluorinated pyrimidines and inhibitors of deoxyuridylate synthesis. Biochem Pharmacol 1982;31:2929–2935.

449. Engstrom PF, MacIntyre JM, Mittelman A, et al. Chemotherapy of advanced colorectal carcinoma. Fluorouracil alone vs two drug combinations using fluorouracil, hydroxyurea, semustine, dacarbazine, razoxane, and mitomycin. A phase III trial by the Eastern Cooperative Oncology Group. Am J Clin Oncol 1984; 7:313–318.

450. Engstrom PF, MacIntyre JM, Schutt AJ, et al. Chemotherapy of large bowel carcinoma—fluorouracil plus hydroxyurea vs methyl-CCNU, oncovin, fluorouracil and streptozotocin. An Eastern Cooperative Oncology Group Study. Am J Clin Oncol 1985;8:358–361.

451. Di Costanzo F, Gasperoni S, Malacarne P, et al. High-dose folinic acid and 5-fluorouracil alone or combined with hydroxyurea in advanced colorectal cancer. A randomized trial of the Italian Oncology Group For Clinical Research. Am J Clin Oncol 1998;21:369–375.

452. Santelli J, Valeriote F. In vivo potentiation of 5-fluorouracil cytotoxicity against AKR leukemia by purines, pyrimidines and their nucleosides and deoxynucleosides. J Natl Cancer Inst 1980;64:69–72.

453. Iigo M, Kuretani K, Hoshi A. Relationship between antitumor effect and metabolites of 5-fluorouracil in combination treatment with 5-fluorouracil and guanosine in ascites sarcoma 180 tumor system. Cancer Res 1983;43:5687–5694.

454. Iigo M, Yamaizumi Z, Nishimura S, et al. Mechanism of potentiation of antitumor activity of 5-fluorouracil by guanine ribonucleotides against adenocarcinoma 755. Eur J Cancer Clin Oncol 1987;23:1059–1065.

455. Parker WB, Klubes P. Enhancement by uridine of the anabolism of 5-fluorouracil in mouse T-lymphoma (S-49) cells. Cancer Res 1985;45:4249–4256.

456. Tuchman M, Ramnaraine ML, O'Dea RF. Effects of uridine and thymidine on the degradation of 5-fluorouracil, uracil and thymine by rat liver dihydropyrimidine dehydrogenase. Cancer Res 1985;45:5553–5556.

457. Fleming RA, Capizzi RL, Muss HB, et al. Phase I study of N-(phosphonacetyl)-l-aspartate with fluorouracil and with or without dipyridamole in patients with advanced cancer. Clin Cancer Res 1996;2:1107–1114.

458. Chu MYW, Naguib FNM, Iltzsch MH, et al. Potentiation of FdUrd antineoplastic activity by the uridine phosphorylase inhibitors benzylacyclouridine and benzyloxybenzylacyclouridine. Cancer Res 1984;44:1852–1856.

459. Darnowski JW, Handschumacher RE. Tissue-specific enhancement of uridine utilization and 5-fluorouracil therapy in mice by benzylacyclouridine. Cancer Res 1985;45:5364–5368.

460. Pu T, Robertson JM, Lawrence TS. Current status of radiation sensitization by fluorinated pyrimidines. Oncology (Basel) 1995;9:707–735.

461. Garrett C, Wataya Y, Santi D. Thymidylate synthetase. Catalysis of dehalogenation of 5-bromo- and 5-iodo-2′- deoxyuridylate. Biochemistry 1979;18:2798.

462. Heidelberger C, Griesback I, Ghobar A. The potentiation of 5-iodo-2′-deoxyuridine of the tumor-inhibitory activity of 5-fluoro-2′-deoxyuridine. Cancer Chemother Rep 1960;6:37.

463. Benson AB, Trump DL, Cummings KB, et al. Modulation of 5-iodo-2′-deoxyuridine metabolism and cytotoxicity in human bladder cancer cells by fluoropyrimidines. Biochem Pharmacol 1985;34:3925.

464. Mancini WR, Stetson PL, Lawrence TS, et al. Variability of 5-bromo-2′-deoxyuridine incorporation into DNA of human glioma cell lines and modulation with fluoropyrimidines. Cancer Res 1991;51:870.

465. Lawrence TS, Davis MA, Maybaum J, et al. Modulation of iododeoxyuridine-mediated radiosensitization by 5-fluorouracil in human colon cancer cells. Int J Radiat Oncol Biol Phys 1992; 22:49.

466. Vazquez-Padua MA, Risueno C, Fischer PH. Regulation of the activation of fluorodeoxyuridine by substrate competition and feedback inhibition in 647V cells. Cancer Res 1989;49:618.

467. Calabresi P, Creasey WA, Prusoff WH, et al. Clinical and pharmacological studies with 5-iodo-2′-deoxyuridine. Cancer Res 1963;23:583.

468. Papac R, Jacobs E, Wong F, et al. Clinical evaluation of the pyrimidine nucleosides 5-fluoro-2′-deoxyuridine and 5- iodo-2′-deoxyuridine. Cancer Chemother Rep 1960;6:143.

469. Young CW, Ellison RR, Sullivan RD, et al. The clinical evaluation of 5-fluorouracil and 5-fluoro-2′-deoxyuridine in solid tumors in adults. Cancer Chemother Rep 1960;6:17.

470. Kinsella TJ, Russo A, Mitchell JB, et al. A phase I study of intravenous iododeoxyuridine as a clinical radiosensitizer. Int J Radiat Oncol Biol Phys 1985;111:1941.

471. Kinsella TJ, Collins J, Rowland J, et al. Pharmacology and phase I/II study of continuous intravenous infusions of iododeoxyuridine and hyperfractionated radiotherapy in patients with glioblastoma multiforme. J Clin Oncol 1988;6:871.

472. Belanger K, Klecker RW Jr, Rowland J, et al. Incorporation of iododeoxyuridine into DNA of granulocytes in patients. Cancer Res 1986;46:6509.

473. Speth PAJ, Kinsella TJ, Chang AE, et al. Selective incorporation of iododeoxyuridine into DNA of hepatic metastases versus normal human liver. Clin Pharmacol Ther 1988;44:369.

474. Speth PAJ, Kinsella TJ, Belanger K, et al. Fluorodeoxyuridine modulation of the incorporation of iododeoxyuridine into DNA of granulocytes. A phase I and clinical pharmacology study. Cancer Res 1988;48:2933.

475. McGinn CJ, Kunugi KA, Tutsch KD, et al. Leucovorin modulation of 5-iododeoxyuridine radiosensitization. A phase I study. Clin Cancer Res 1986;2:1299–1305.

476. Remick SC, Benson AB, Weese JL, et al. Phase I trial of hepatic artery infusion of 5-iodo-2′-deoxyuridine and 5-fluorouracil in patients with advanced hepatic malignancy. Biochemically based combination therapy. Cancer Res 1989;49:6437.

477. Pastor-Anglada M, Felipe A, Casado FJ. Transport and mode of action of nucleoside derivatives used in chemical and antiviral therapies. Trends Pharmacol Sci 1998;19:424-40.

478. Grem JL, Fischer PH. Augmentation of 5-fluorouracil cytotoxicity in human colon cancer cells by dipyridamole. Cancer Res 1985;45:2967–2972.

479. Grem JL, Fischer PH. Modulation of fluorouracil metabolism and cytotoxicity by nitrobenzylthioinosine. Biochem Pharmacol 1986;35:2651–2654.

480. Grem JL, Fischer PH. Alteration of fluorouracil metabolism in human colon cancer cells by dipyridamole with a selective increase in fluorodeoxyuridine monophosphate levels. Cancer Res 1986;46:6191–6199.

481. Budd GT, Jayaraj A, Grabowski D, et al. Phase I trial of dipyridamole with 5-fluorouracil and folinic acid. Cancer Res 1990; 50:7206–7211.

482. Fischer PH, Willson JKV, Risueno C, et al. Biochemical assessment of the effects of acivicin and dipyridamole given as a continuous 72-hour intravenous infusion. Cancer Res 1988;48: 5591–5596.

483. Bailey H, Wilding G, Tutsch KD, et al. A phase I trial of 5- fluorouracil, leucovorin, and dipyridamole given by concurrent 120-h continuous infusions. Cancer Chemother Pharmacol 1992;30:297–302.

484. Willson JKV, Fischer PH, Remick SC, et al. Methotrexate and dipyridamole combination chemotherapy based upon inhibition of nucleoside salvage in humans. Cancer Res 1989;49:1866–1889.

485. Köhne C-H, Hiddemann W, Schüller J, et al. Failure of orally administered dipyridamole to enhance the antineoplastic activity of fluorouracil in combination with leucovorin in patients with advanced colorectal cancer. A prospective randomized trial. J Clin Oncol 1995;13:1201–1208.

486. Scanlon KJ, Newman EM, Lu Y, et al. Biochemical basis for cisplatin and 5-fluorouracil synergism in human ovarian carcinoma cells. Proc Natl Acad Sci U S A 1986;83:8923–8925.

487. Pratesi G, Gianni L, Manzotti C, et al. Sequence dependence of the antitumor and toxic effects of 5-fluorouracil and cis-diamminedichloroplatinum combination on primary colon tumors in mice. Cancer Chemother Pharmacol 1988;20: 237–241.

488. Esaki T, Nakano S, Tatsumoto T, et al. Inhibition by 5-fluorouracil of cis-diamminedichloroplatinum(II)-induced DNA interstrand cross-link removal in a HST-1 human squamous carcinoma cell line. Cancer Res 1992;52:6501–6506.

489. Tsai C-M, Hsiao S-H, Frey CM, et al. Combination cytotoxic effects of cis-diamminedichloroplatinum(II) and 5- fluorouracil with and without leucovorin against human non-small cell lung cancer cell lines. Cancer Res 1993; 53:1079–1084.

490. Johnston PG, Geoffroy F, Drake J, et al. The cellular interaction of 5-fluorouracil and cisplatin in a human colon carcinoma cell line. Eur J Cancer 1996;32A:2148–2154.

491. Araki H, Fukushima M, Kamiyama Y, Shirasaka T. Effect of consecutive lower-dose cisplatin in enhancement of 5-fluorouracil cytotoxicity in experimental tumor cells in vivo. Cancer Lett 200;160:185–191.

492. Shirasaka T, Shimamoto Y, Ohshimo H, et al. Metabolic basis of the synergistic antitumor activities of 5fluorouracil and cisplatin in rodent tumor cells models in vivo. Cancer Chemother Pharmacol 1993;32:167–172.

493. Lokich JJ, Ahlgren HD, Cantrell J, et al. A prospective randomized comparison of protracted infusional 5-fluorouracil with or without weekly bolus cisplatin in metastatic colorectal carcinoma. Cancer 1991;67:14–19.

494. Diaz-Rubio E, Jimeno J, Anton A, et al. A prospective randomized trial of continuous infusion 5-fluorouracil (5-FU) versus 5-FU plus cisplatin in patients with advanced colorectal cancer. A trial of the Spanish Cooperative Group for Digestive Tract Tumor Therapy (T.T.D.). Am J Clin Oncol 1992;15:56–60.

495. Hansen RM, Ryan L, Anderson T, et al. Phase III study of bolus versus infusion fluorouracil with or without cisplatin in advanced colorectal cancer. J Natl Cancer Inst 1996;88:668–674.

496. Raymond E, Buquet-Fagot F, Djelloul C, et al. Antitumor activity of oxaliplatin in combination with 5-fluorouracil and the thymidylate synthase inhibitor AG337 in human colon, breast and ovarian cancers. Anticancer Drugs 1997;8:876–885.

497. Fischel JL, Etienne MC, Formento P, et al. Search for the optimal schedule for the oxaliplatin/5-fluorouracil association modulated or not by folinic acid. Preclinical data. Clin Cancer Res 1998;4:2529–2535.

498. Fischel JL, Formento P, Ciccolini J, et al. Impact of the oxaliplatin-5-luorouracil-folinic acid combination on respective intracellular determinants of drug activity. Br J Cancer 2002;86:1162–1168.

499. Yeh K-H, Cheng A-L, Wan J-P, et al. Down-regulation of thymidylate synthase expression and its steady-state mRNA by oxaliplatin in colon cancer cells. Anti-Cancer Drugs 2004;15: 371–376.

500. de Gramont A, Figer A, Seymour M, et al. Leucovorin and fluorouracil with or without oxaliplatin as first-line treatment in advanced colorectal cancer. J Clin Oncol 2000; 18:2938–2947.

501. Andre T, Boni C, Mounedji-Boudiaf L, Navarro M, Tabernero J, Hickish T, et al. Oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment for colon cancer. N Engl J Med 2004; 350:2343–2351.

502. Kano Y, Akutsu M, Tsunoda S, et al. Schedule-dependent interaction between paclitaxel and 5-fluorouracil in human carcinoma cell lines in vitro. Br J Cancer 1996;74:704–710.

503. Johnson KR, Wang L, Miller MC III, et al. 5-Fluorouracil interferes with paclitaxel cytotoxicity against human solid tumor cells. Clin Cancer Res 1997;3:1739–1745.

504. Grem JL, Nguyen D, Monahan BP, et al. Sequence-dependent antagonism between fluorouracil and paclitaxel in human breast cancer cells. Biochem Pharmacol 1999;58:477–486.

505. Houghton JA, Cheshire PJ, Hallman II JD, et al. Evaluation of irinotecan in combination with 5-fluorouracil or etoposide in xenograft models of colon adenocarcinoma and rhabdomyosarcoma. Clin Cancer Res 1996;2:107–118.

506. Guichard S, Cussac D, Hennebelle I, et al. Sequence-dependent activity of the irinotecan-5FU combination in human colon-cancer model HT-29 in vitro and in vivo. Int J Cancer 1997; 73:729–734.

507. Aschele C, Baldo C, Sobrero AF, et al. Schedule-dependent synergism between raltitrexed and irinotecan in human colon cancer cells in vitro. Clin Cancer Res 1998;4: 1323–1330.

508. Pavillard V, Formento P, Rostagno P, et al. Combination of irinotecan (CPT11) and 5-fluorouracil with an analysis of cellular determinants of drug activity. Biochem Pharmacol 1998; 56:1315–1322.

509. Mans DR, Grivicich I, Peters GJ, Schwartsmann G. Sequence-dependent growth inhibition and DNA damage formation by the irinotecan-5-fluorouracil combination in human colon carcinoma cell lines. Eur J Cancer 1999;35:1851–1861.

510. Azrak RG, Cao S, Slocum HK, et al. Therapeutic synergy between irinotecan and 5-fluorouracil against human tumor xenografts. Clin Cancer Res 2004;10:1121–1129.

511. Heidelberger C, Griesvach L, Montag BJ, et al. Studies on fluorinated pyrimidines. II. Effects on transplanted tumors. Cancer Res 1958;18:305–317.

512. Byfield JE, Calabro-Jones P, Klisak I, et al. Pharmacologic requirements for obtaining sensitization of human tumor cells in vitro to combined 5-fluorouracil or ftorafur and x-rays. Int J Radiat Oncol Biol Phys 1982;8:1923–1933.

513. Weinberg MJ, Rauth AM. 5-Fluorouracil infusions and fractionated doses of radiation. Studies with a murine squamous cell carcinoma. Int J Radiat Oncol Biol Phys 1987;13:1691–1699.

514. Ishikawa T, Tanaka Y, Ishitsuka H, et al. Comparative antitumor activity of 5-fluorouracil and 5′-deoxyfluorouridine in combination with radiation therapy in mice bearing colon 26 adenocarcinoma. Cancer Res 1989;80:583–591.

515. Smalley SR, Kimler BF, Evans RG. 5-Fluorouracil modulation of radiosensitivity in cultured human carcinoma cells. Int J Radiat Oncol Biol Phys 1991;20:207–211.

516. Bruso CE, Shewach DS, Lawrence TS. Fluorodeoxyuridine- induced radiosensitization and inhibition of DNA double- strand break repair in human colon cancer cells. Int J Radiat Oncol Biol Phys 1990;19:1411–1417.

517. Lawrence T, Heimburger D, Shewach DL. The effects of leucovorin and dipyridamole on fluoropyrimidine-induced radiosensitization. Int J Radiat Oncol Biol Phys 1991;20:377–381.

518. Miller EM, Kinsella TJ. Radiosensitization by fluorodeoxyuridine. Effects of thymidylate synthase inhibition and cell synchronization. Cancer Res 1992;52:1687–1694.

519. Davis MA, Tang HY, Maybaum J, et al. Dependence of fluorodeoxyuridine-mediated radiosensitization on S phase progression. Int J Radiat Biol 1995;67:509–517.

520. Lawrence TS, Davis MA, Loney TL. Fluoropyrimidine- mediated radiosensitization depends on cyclin E-dependent kinase activation. Cancer Res 1996;56:3203–3206.

521. Lawrence TS, Tepper JE, Blackstock AW. Fluoropyrimidine- radiation interactions in cells and tumors. Semin Radiat Oncol 1997;7:260–266.

522. Bartelink H, Roelofsen F, Eschwege F, et al. Concomitant radiotherapy and chemotherapy is superior to radiotherapy alone in the treatment of locally advanced anal cancer. Results of a phase III randomized trial of the European Organization for Research and Treatment of Cancer Radiotherapy and Gastrointestinal Cooperative Groups. J Clin Oncol 1997;15: 2040–2049.

523. Morris M, Eifel PJ, Lu J, et al. Pelvic radiation with concurrent chemotherapy compared with pelvic and para-aortic radiation for high-risk cervical cancer. N Engl J Med 1999;340: 1137–1143.

524. Cooper JS, Guo MD, Herskovic A, et al. Chemoradiotherapy of locally advanced esophageal cancer. Long-term follow-up of a prospective randomized trial (RTOG 85-01). Radiation Therapy Oncology Group. JAMA 1999;281:1623–1627.

525. O'Connell MJ, Martenson JA, Wieand HS, et al. Improving adjuvant therapy for rectal cancer by combining protracted infusion fluorouracil with radiation therapy after curative surgery. N Engl J Med 1994;33:502–507.

526. Au JL, Sadee W. The pharmacology of ftorafur. Recent Results Cancer Res 1981;76:100–114.

527. Benvenuto JA, Liehr JG, Winkler T, et al. Human urinary metabolites of 1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur). Cancer Res 1980;40:2814–3870.

528. El Sayed YM, Sadee W. Metabolic activation of ftorafur. the microsomal oxidative pathway. Biochem Pharmacol 1982;31: 3006–3008.

529. El Sayed YM, Sadee W. Metabolic activation of R, S-1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur) to 5-fluorouracil by soluble enzymes. Cancer Res 1983;43:4039–4044.

530. Komatsu T, Yamazaki H, Shimada N, et al. Roles of cytochrome P450 1A2, 2A6, and 2C8 in 5-fluorouracil formation from tegafur, an anti-cancer prodrug, in human liver microsomes. Drug Metab Dispos 2000;28:1457–1463.

531. Tomoko Komatsu, Hiroshi Yamazaki, Noriaki Shimada, et al. Involvement of microsomal cytochrome P450 and cytosolic thymidine phosphorylase in 5-fluorouracil formation from tegafur in human liver. Clin Cancer Res 2001;7:675–681.

532. Fuji S, Ikenaka K, Fukushima M, et al. Effect of uracil and its derivatives on antitumor activity of 5-fluorouracil and 1- (2-tetrahydrofuryl)-5-fluorouracil. Jpn J Cancer Res 1979; 69:763–772.

533. Benvenuto J, Lu K, Hall SW, et al. Metabolism of 1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur). Cancer Res 1978;38:3867–3870.

534. Hall SW, Valdivieso M, Benjamin RS. Intermittent high single dose ftorafur. A phase I clinical trial with pharmacologic- toxicity correlations. Cancer Treat Rep 1977;61:1495–1498.

535. Antilla MI, Sotaniemi EA, Kaiaralcoma MI, et al. Pharmacokinetics of ftorafur after intravenous and oral administration. Cancer Chemother Pharmacol 1983;10:150–153.

536. Hornbeck CL, Griffiths JC, Floyd RA, et al. Serum concentrations of 5-FU, ftorafur, and a major serum metabolite following ftorafur chemotherapy. Cancer Treat Rep 1981;65:69–72.

537. Blokhina NG, Vozny EK, Garin AM. Results of treatment of malignant tumors with ftorafur. Cancer 1972;30:390–392.

538. Friedman MA, Ignoffo RJ. A review of the United States clinical experience of the fluoropyrimidine, ftorafur (NSC 148958). Cancer Treat Rev 1980;7:205–213.

539. Ansfield FJ, Kallas GJ, Singson JP. Phase I–II studies of oral tegafur (ftorafur). J Clin Oncol 1983;1:107–110.

540. Kajanti MJ, Pyrhönen SO, Maiche AG. Oral tegafur in the treatment of metastatic breast cancer. A phase II study. Eur J Cancer 1993;29A:863–866.

541. Palmeri S, Gebbia V, Russo A, et al. Oral tegafur in the treatment of gastrointestinal tract cancers. A phase II study. Br J Cancer 1990;61:475–478.

542. Bjerkeset T, Fjosne HE. Comparison of oral ftorafur and intravenous 5-fluorouracil in patients with advanced cancer of the stomach, colon or rectum. Oncology 1986;43:212–215.

543. Manzuik LV, Perevodchikova NI, Gorbunova VA, et al. Initial clinical experience with oral ftorafur and oral 6R, S-leucovorin in advanced colorectal carcinoma. Eur J Cancer 1993;29A: 1793–1794.

544. Tang SG, Hornbeck CL, Byfield JE. Enhanced accumulation of 5-fluorouracil in human tumors in athymic mice by co-administration of ftorafur and uracil. Int J Radiat Oncol Biol Phys 1984;10:1687–1689.

545. Ho DH, Cobington WP, Pazdur R, et al. Clinical pharmacology of combined oral uracil and ftorafur. Drug Metab Dispos 1992;20:936–940.

546. Fujita K, Munakata A. Concentration of 5-fluorouracil in renal cells from cancer patients administered a mixture of 1- (2-tetrahydrofuryl)-5-fluorouracil and uracil. Int J Clin Pharmacol Res 1991;11:171–174.

547. Takayama H, Konami T, Konishi T, et al. Studies on 5-FU concentration in serum and bladder tumor tissue after oral administration of UFT. Acta Urol Jpn 1986;32:1449–1453.

548. Basaki Y, Chikahisa L, Aoyagi K, et al. gamma-Hydroxybutyric acid and 5-fluorouracil, metabolites of UFT, inhibit the angiogenesis induced by vascular endothelial growth factor. Angiogenesis 2001;4(3):163–173.

549. Ota K, Taguchi T, Kimura K. Report on nationwide pooled data and cohort investigation in UFT phase II study. Cancer Chemother Pharmacol 1988;22:333–338.

550. Ho DH, Pazdur R, Covington W, et al. Comparison of 5-fluorouracil pharmacokinetics in patients receiving continuous 5-fluorouracil infusion and oral uracil plus N1-(2′-tetrahydrofuryl)-5-fluorouracil. Clin Cancer Res 1998;4:2085–2088.

551. Muggia FM, Wu X, Spicer D, et al. Phase I and pharmacokinetic study of oral UFT, a combination of the 5-fluorouracil prodrug tegafur and uracil. Clin Cancer Res 1996;2:1461–1467.

552. Pazdur R, Lassere Y, Rhodes V, et al. Phase II trial of uracil and tegafur plus oral leucovorin. An effective oral regimen in the treatment of metastatic colorectal carcinoma. J Clin Oncol 1994;12:2296–2300.

553. Gonzalez Baron M, Feliu J, Garcia Giron C, et al. UFT modulated with leucovorin in advanced colorectal cancer. Oncopaz experience. Oncology 1997;54:24–29.

554. Ichikura T, Tomimatsu S, Okusa Y, et al. Thymidylate synthase inhibition by an oral regimen consisting of tegafur- uracil (UFT) and low-dose leucovorin for patients with gastric cancer. Cancer Chemother Pharmacol 1996;38:401–405.

555. Meropol NJ, Sonnichsen DS, Birkhofer MJ, et al. Bioavailability and phase II study of oral UFT plus leucovorin in patients with relapsed or refractory colorectal cancer. Cancer Chemother Pharmacol 1999;43:221–226.

556. Sulkes A, Benner SE, Canetta RM. Uracil-ftorafur. An oral fluoropyrimidine active in colorectal cancer. J Clin Oncol 1998;16: 3461–3475.

557. Carmichael J, Tadeusz P, Radstone D, et al. Randomized comparative study of tegafur/uracil and oral leucovorin versus parenteral fluorouracil and leucovorin in patients with previously untreated metastatic colorectal cancer. J Clin Oncol 2002;20: 3617–3627.

558. Douillard J-Y, Hoff PM, Skillings JR, et al. Multicenter phase III study of uracil/tegafur and oral leucovorin versus fluorouracil and leucovorin in patients with previously untreated metastatic colorectal cancer. J Clin Oncol 2002;20:3605–3616.

559. Eda H, Fujimoto K, Watanabe S-I, et al. Cytokines induce uridine phosphorylase in mouse colon 26 carcinoma cells and make the cells more susceptible to 5′-deoxy-5-fluorouridine. Jpn J Cancer Res 1993;84:341–347.

560. Eda H, Fujimoto K, Watanabe S-I, et al. Cytokines induce thymidine phosphorylase in tumor cells and make the cells more susceptible to 5′-deoxy-5-fluorouridine. Cancer Chemother Pharmacol 1993;32:333–339.

561. Armstrong RD, Cadman E. 5′-Deoxyfluorouridine selective toxicity for human tumor cells compared to human bone marrow. Cancer Res 1983;43:2525–2528.

562. Miwa M, Nishimura J, Kayamiyama T, et al. Conversion of 5′-deoxyuridine to 5-FU by pyrimidine nucleoside phosphorylase in normal and tumor tissues from rodents bearing tumors and cancer patients. Jpn J Cancer Chemother 1987;14:2924–2929.

563. Peters GJ, Braakhuis BJM, de Bruijn EA, et al. Enhanced therapeutic efficacy of 5′-deoxy-5-fluorouridine in 5-fluorouracil resistant head and neck tumours in relation to 5-fluorouracil metabolising enzymes. Br J Cancer 1989;59:327–334.

564. Nio Y, Kimura H, Tsubone M, et al. Antitumor activity of 5′-deoxy-5-fluorouridine in human digestive organ cancer xenografts and pyrimidine nucleoside phosphorylase activity in normal and neoplastic tissues from human digestive organs. Anticancer Res 1992;12:1141–1146.

565. el Khouni MH, el Kouni MM, Naguib NM. Differences in activities and substrate specificity of human and murine pyrimidine nucleoside phosphorylases. Implications for chemotherapy with 5 fluoropyrimidines. Cancer Res 1993;53:3687–3693.

566. el Khouni MH, Naguib FNM, Chu SH, et al. Effect of the n-glycosidic bond conformation and modifications in the pentose moiety on the binding of nucleoside ligands to urdidine phosphorylase. Mol Pharmacol 1988;34:104–110.

567. Veres Z, Szabolcs A, Szinai I, et al. Enzymatic cleavage of 5- substituted-2′-deoxyuridines by pyrimidine nucleoside phosphorylases. Biochem Pharmacol 1986;35:1057–1059.

568. Vertongen F, Fondu P, Van den Heule B, et al. Thymidine kinase and thymidine phosphorylase activities in various types of leukemia and lymphoma. Tumour Biol 1984;5:303–311.

569. de Bruijn EA, van Oosterom AT, Tjaden UR, et al. Pharmacology of 5′-deoxy-5-fluorouridine in patients with resistant ovarian cancer. Cancer Res 1985;45:5931–5935.

570. Schaaf LJ, Dobbs BR, Edwards IR, et al. The pharmacokinetics of doxifluridine and 5-fluorouracil after single intravenous infusions of doxifluridine to patients with colorectal cancer. Eur J Clin Pharmacol 1988;34:439–443.

571. Malet-Martino MC, Servin P, Bernadou J, et al. Human urinary excretion of doxifluoridine and metabolites during a 5-day chemotherapeutic schedule using fluorine-19 nuclear magnetic resonance spectrometry. Invest New Drugs 1987;5:273–279.

572. Martino R, Bernadou J, Malet-Martino MC, et al. Excretion of doxifluridine catabolites in human bile assessed by 19F NMR spectrometry. Biomed Pharmacother 1987;41:104–106.

573. Reece PA, Olver IN, Morris RG, et al. Pharmacokinetic study of doxifluridine given by 5-day stepped-dose infusion. Cancer Chemother Pharmacol 1990;25:274–278.

574. Abele R, Alberto P, Seematter RJ, et al. Phase I clinical study with 5′-deoxy-5′-fluorouridine, a new fluoropyrimidine derivative. Cancer Treat Rep 1982;1307–1313.

575. Hurteloup P, Armand JP, Cappelaere P, et al. Phase II clinical evaluation of doxifluridine. Cancer Treat Rep 1986;70:1339–1340.

576. Fossa SD, Dahl O, Hoel R, et al. Doxifluridine (5′dFUR) in patients with advanced colorectal carcinoma. Cancer Chemother Pharmacol 1985;15:161–163.

577. Alberto P, Mermillod B, Germano G, et al. A randomized comparison of doxifluridine and fluorouracil in colorectal cancer. Eur J Cancer Clin Oncol 1998;24:559–563.

578. Alberto P, Jungi WF, Siegenthaler P, et al. A phase II study of doxifluridine in patients with advanced breast cancer. Eur J Cancer Clin Oncol 1988;24:565–566.

579. Heier MS, Fossa SD. Wernicke-Korsakoff-like syndrome in patients with colorectal carcinoma treated with high-dose doxifluridine. Acta Neurol Scand 1986;73:449–457.

580. Schuster D, Heim ME, Dombernowski P, et al. Prospective randomized phase III trial of doxifluridine versus 5-fluorouracil in patients with advanced colorectal cancer. Onkologie 1991;14: 333–337.

581. Bajetta E, Colleoni M, Rosso R, et al. Prospective randomised trial comparing fluorouracil versus doxifluridine for the treatment of advanced colorectal cancer. Eur J Cancer 1993;29A: 1658–2663.

582. Alberto P, Winkelmann JJ, Paschoud N, et al. Phase I study of oral doxifluridine using two schedules. Eur J Cancer Clin Oncol 1989;25:905–908.

583. Bajetta E, Colleoni M, Di Bartolomeo M, et al. Doxifluridine and leucovorin. An oral treatment combination in advanced colorectal cancer. J Clin Oncol 1995;13:2613–2619.

584. Dooley M, Goa KL. Capecitabine. Drugs 1999;58(1):69–76.

585. Tabata T, Katoh M, Tokudome S, et al. Bioactivation of capecitabine in human liver: involvement of the cytosolic enzyme on 5′-deoxy-5-fluorocytidine formation. Drug Metab Dispos 2004;32(7):762–767.

586. Tabata T, Katoh M, Tokudome S, et al. Identification of the cytosolic carboxylesterase catalyzing the 5′-deoxy-5-fluorocytidine formation from capecitabine in human liver. Drug Metab Dispos 2004 32():1103–1110.

587. Budman DR, Meropol NJ, Reigner B, et al. Preliminary studies of a novel oral fluoropyrimidine carbamate. Capecitabine. J Clin Oncol 1998;16:1795–1802.

588. Mackean M, Planting A, Twelves C, et al. Phase I and pharmacologic study of intermittent twice-daily oral therapy with capecitabine in patients with advanced and/or metastatic cancer. J Clin Oncol 1998;16:2977–2985.

589. Judson IR, Beale PJ, Trigo JM, et al. A human capecitabine excretion balance and pharmacokinetic study after administration of a single oral dose of 14C-labelled drug. Invest New Drugs 1999;17(1):49–56.

590. Miwa M, Ura M, Nishida M, et al. Design of a novel oral fluoropyrimidine carbamate, capecitabine, which generates 5-fluorouracil selectively in tumours by enzymes concentrated in human liver and cancer tissue. Eur J Cancer 1998;34:1274–1281.

591. Ishikawa T, Utoh M, Sawada N, et al. Tumor selective delivery of 5-fluorouracil by capecitabine, a new oral fluoropyrimidine carbamate, in human cancer xenografts. Biochem Pharmacol 1998;55:1091–1097.

592. Ishikawa T, Sekiguchi F, Fukase Y, et al. Positive correlation between the efficacy of capecitabine and doxifluridine and the ratio of thymidine phosphorylase to dihydropyrimidine dehydrogenase activities in tumors in human cancer xenografts. Cancer Res 1998;58:685–690.

593. Tsukamoto Y, Kato Y, Ura M, et al. Investigation of 5-FU disposition after oral administration of capecitabine, a triple-prodrug of 5-FU, using a physiologically based pharmacokinetic model in a human cancer xenograft model: comparison of the simulated 5-FU exposures in the tumour tissue between human and xenograft model. Biopharm Drug Dispos 2001;22(1):1–14.

594. Chung YL, Troy H, Judson IR, et al. Noninvasive measurements of capecitabine metabolism in bladder tumors overexpressing thymidine phosphorylase by fluorine-19 magnetic resonance spectroscopy. Clin Cancer Res 2004;10:3863–3870.

595. Mori K, Hasegawa M, Nishida M, et al. Expression levels of thymidine phosphorylase and dihydropyrimidine dehydrogenase in various human tumor tissues. Int J Oncol 2000;17: 33–38.

596. Cassidy J, Dirix L, Bissett D, et al. A Phase I study of capecitabine in combination with oral leucovorin in patients with intractable solid tumors. Clin Cancer Res 1998;4:2755–2761.

597. Saif MW, Quinn MG, Thomas RR, et al. Cardiac toxicity associated with capecitabine therapy. Acta Oncol 2003;42:342–344.

598. Walkhorm B, Fraunfelder FT. Severe ocular irritation and corneal deposits associated with capecitabine use. New Eng J Med 2004;343:740–741.

599. Couch LS, Groteluschen DL, Stewart JA, et al. Capecitabine-related neurotoxicity presenting as trismus. Clin Colorectal Cancer 2003;3:121–123.

600. Schuller J, Cassidy J, Dumont E, et al. Preferential activation of capecitabine in tumor following oral administration to colorectal cancer patients. Cancer Chemother Pharmacol 2000;45: 291–297

601. Nishimura G, Terada I, Kobayashi T, et al. Thymidine phosphorylase and dihydropyrimidine dehydrogenase levels in primary colorectal cancer show a relationship to clinical effects of 5′-deoxy-5-fluorouridine as adjuvant chemotherapy. Oncol Rep 2002;9:479–482.

602. Reigner B, Blesch K, Weidekamm E. Clinical pharmacokinetics of capecitabine. Clin Pharmacokinet 2001;40:85–104.

603. Reigner B, Verweij J, Dirix L, et al. Effect of food on the pharmacokinetics of capecitabine and its metabolites following oral administration in cancer patients. Clin Cancer Res 1998;4: 941–948.

604. Twelves C, Glynne-Jones R, Cassidy J, et al. Effect of hepatic dysfunction due to liver metastases on the pharmacokinetics of capecitabine and its metabolites. Clin Cancer Res 1999;5: 1696–1702.

605. Poole C, Gardiner J, Twelves C, et al. Effect of renal impairment on the pharmacokinetics and tolerability of capecitabine (Xeloda) in cancer patients. Cancer Chemother Pharmacol 2002;49:225–234.

606. Xu Y, Grem JL. Liquid chromatography-mass spectrometry method for the analysis of the anti-cancer agent capecitabine and its nucleoside metabolites in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci 2003;783(1):273–285.

607. Zufia L, Aldaz A, Giraldez J. Simple determination of capecitabine and its metabolites by liquid chromatography with ultraviolet detection in a single injection. J Chromatogr B Analyt Technol Biomed Life Sci 2004;809:51–58.

608. van Laarhoven HW, Klomp DW, Kamm YJ, et al. In vivo monitoring of capecitabine metabolism in human liver by 19fluorine magnetic resonance spectroscopy at 1.5 and 3 Tesla field strength. Cancer Res 2003;63:7609–7612.

609. Mader RM, Schrolnberger C, Rizovski B, et al. Penetration of capecitabine and its metabolites into malignant and healthy tissues of patients with advanced breast cancer. Br J Cancer 2003;88:782–787.

610. Hoff PM, Ansari R, Batist G, et al. Comparison of oral capecitabine versus intravenous fluorouracil plus leucovorin as first-line treatment in 605 patients with metastatic colorectal cancer: results of a randomized phase III study. J Clin Oncol 2001;19:2282–2292.

611. Van Cutsem E, Twelves C, Cassidy J, et al. Oral capecitabine compared with intravenous fluorouracil plus leucovorin in patients with metastatic colorectal cancer: results of a large phase III study. J Clin Oncol 2001;19:4097–4106

612. Endo M, Shinbori N, Fukase Y, et al. Induction of thymidine phosphorylase expression and enhancement of efficacy of capecitabine or 5′-deoxy-5-fluorouridine by cyclophosphamide in mammary tumor models. Int J Cancer 1999;83: 127–134.

613. Blanquicett C, Gillespie GY, Nabors LB, et al. Induction of thymidine phosphorylase in both irradiated and shielded, contralateral human U87MG glioma xenografts: implications for a dual modality treatment using capecitabine and irradiation. Mol Cancer Ther 2002;1:1139–1145.

614. Xiao YS, Tang ZY, Fan J, et al. Interferon-alpha 2a up-regulated thymidine phosphorylase and enhanced antitumor effect of capecitabine on hepatocellular carcinoma in nude mice. J Cancer Res Clin Oncol 2004.

615. Magne N, Fischel JL, Dubreuil A, et al. ZD1839 (Iressa) modifies the activity of key enzymes linked to fluoropyrimidine activity: rational basis for a new combination therapy with capecitabine. Clin Cancer Res 2003;9:4735–4742.

616. Porter DJ, Chestnut WG, Merrill BM, et al. Mechanism- based inactivation of dihydropyrimidine dehydrogenase by 5-ethynyluracil. J Biol Chem 1992;267:5236–5242.

617. Spector T, Harrington JA, Porter DJ. 5-Ethynyluracil (776C85). Inactivation of dihydropyrimidine dehydrogenase in vivo. Biochem Pharmacol 1993;46:2243–2248.

618. Baccanari DP, Davis ST, Knick VC, et al. 5-Ethynyluracil (776C85). A potent modulator of the pharmacokinetics and antitumor efficacy of 5-fluorouracil. Proc Natl Acad Sci U S A 1993;90:11064–11068.

619. Cao S, Rustum YM, Spector T. 5-Ethynyluracil (776C85). Modulation of 5-fluorouracil efficacy and therapeutic index in rats bearing advanced colorectal carcinoma. Cancer Res 1994; 54:1507–1510.

620. Spector T, Cao S, Rustum YM, et al. Attenuation of the antitumor activity of 5-fluorouracil by (R)-5-fluoro-5,6- dihydrouracil. Cancer Res 1995;55:1239–1241.

621. Arellano M, Malet-Martino M, Martino R, et al. 5-Ethynyluracil (GW776). Effects on the formation of the toxic catabolites of 5-fluorouracil, fluoroacetate and fluorohydroxypropionic acid in the isolated perfused rat liver model. Br J Cancer 1997;76: 1170–1180.

622. Adams ER, Leffert JJ, Craig DJ, et al. In vivo effect of 5- ethynyluracil on 5-fluorouracil metabolism determined by 19F nuclear magnetic resonance spectroscopy. Cancer Res 1999;59:122–127.

623. Schilsky RL, Hohneker J, Ratain MJ, et al. Phase I clinical and pharmacologic study of eniluracil plus fluorouracil in patients with advanced cancer. J Clin Oncol 1998;16:1450–1457.

624. Baker SD, Khor SP, Adjei AA, et al. Pharmacokinetic, oral bioavailability, and safety study of fluorouracil in patients treated with 776C85, an inactivator of dihydropyrimidine dehydrogenase. J Clin Oncol 1996;14:3085–3096.

625. Mani S, Hochster H, Beck T, et al. Multicenter phase II study to evaluate a 28-day regimen of oral fluorouracil plus eniluracil in the treatment of patients with previously untreated metastatic colorectal cancer. J Clin Oncol 2000;18:2894–2901.

626. Smith IE, Johnston SR, O'Brien ME, et al. Low-dose oral fluorouracil with eniluracil as first-line chemotherapy against advanced breast cancer: a phase II study. J Clin Oncol 2000; 18:2378–2384.

627. Schilsky RL, Levin J, West WH, et al. Randomized, open-label, phase III study of a 28-day oral regimen of eniluracil plus fluorouracil versus intravenous fluorouracil plus leucovorin as first-line therapy in patients with metastatic/advanced colorectal cancer. J Clin Oncol 2002;20:1519–1526.

628. Grem JL, Harold N, Shapiro J, et al. Phase I and pharmacokinetic trial of weekly oral fluorouracil given with eniluracil and low-dose leucovorin to patients with solid tumors. J Clin Oncol 2000;18:3952–3963.

629. Keith B, Guo XD, Zentko S, et al. Impact of two weekly schedules of oral eniluracil given with fluorouracil and leucovorin on the duration of dihydropyrimidine dehydrogenase inhibition. Clin Cancer Res 2002;8:1045–1050

630. Shirasaka T, Shimamato Y, Ohshimo H, et al. Development of a novel form of an oral 5-fluorouracil derivative (S-1) directed to the potentiation of the tumor selective cytotoxicity of 5-fluorouracil by two biochemical modulators. Anticancer Drugs 1996;7:548–557.

631. Fujii S, Fukushima M, Shimamoto Y, et al. Antitumor activity of BOF-A2, a new 5-fluorouracil derivative. Jpn J Cancer Res 1989;80:173–181

632. Schöfski P. The modulated oral fluoropyrimidine prodrug S-1, and its use in gastrointestinal cancer and other solid tumors. Anti-Cancer Drugs 2004;15:85–106.

633. Sakamoto J, Kodaira S, Hamada C, et al. An individual patient data meta-analysis of long supported adjuvant chemotherapy with oral carmofur in patients with curatively resected colorectal cancer. Oncol Rep 2001;8:697–703.

634. Rich TA, Shepard RC, Mosley ST. Four decades of continuing innovation with fluorouracil: current and future approaches to fluorouracil chemoradiation therapy. J Clin Oncol 2004;22:2214–2232.

635. Masci G, Magagnoli M, Zucali PA, et al. Minidose warfarin prophylaxis for catheter-associated thrombosis in cancer patients: can it be safely associated with fluorouracil-based chemotherapy? J Clin Oncol 2003;21:736–739.

636. Gilbar PJ, Brodribb TR. Phenytoin and fluorouracil interaction. Ann Pharmacother 2001;35:1367–1370.

637. Afsar A, Lee C, Riddick DS. Modulation of the expression of constitutive rat hepatic cytochrome P450 isozymes by 5-fluorouracil. Can J Physiol Pharmacol 1996;74:150–156.

638. Zhou Q, Chan E. Effect of 5-fluorouracil on the anticoagulant activity and the pharmacokinetics of warfarin enantiomers in rats. Eur J Pharm Sci 2002;17:73-80.

639. Konishi H, Yoshimoto T, Morita K, et al. Depression of phenytoin metabolic capacity by 5-fluorouracil and doxifluridine in rats. J Pharm Pharmacol 2003;55:143-149.