Fluorouracil (5-FU) has been utilized
extensively as a single agent or in combination with other agents in
the clinical management of gastrointestinal malignancies and
carcinomas of the breast and ovary, and squamous carcinomas of the
head and neck.[1,2] Administered as a single agent, 5-FU is generally
considered the standard therapy for patients with adenocarcinoma of
the colon; however, the drug produces a response rate of < 20% in
this disease, with a median survival of < 9 months. Because of
the wide spectrum of marginal antitumor activity exhibited by 5-FU,
numerous investigators are trying to enhance the antitumor activity
and therapeutic selectivity of the compound by combining it with
other agents, eg, calcium folinate.
To exert its antitumor activity, 5-FU must enter the target cells and
interfere with one of two pathways: 1) metabolism to 5-fluorodeoxyuridine
monophosphate (FdUMP), a potent inhibitor of the target enzyme
thymidylate synthase (dTMPS)[3,4]; or 2) metabolism to
5-fluorouridine triphosphate (FUTP), which is subsequently
incorporated into cellular ribonucleic acid (RNA) in place of the
normal metabolite, yielding fraudulent RNA[5,6] (Figure
1). The relative importance of each pathway to the antitumor
activity of 5-FU varies with different tumors.
Calcium folinate is rapidly metabolized to various cofactors with
6R-LV, 5-LV, and 5-methyltetrahydrofolate (5-CH3FH4)
being the dominant plasma cofactors. Significant differences in
elimination plasma half-life exist among these metabolites. Because
it has been demonstrated that both 5-LV and 5-CH3FH4
can modulate the cytotoxicity of 5-FU, it is not known whether each
alone or both of these active metabolites play a major role in the
intracellular accumulation of 5,10-methylenetetrahydrofolate (N5,N10CH2FH4).
The concept of modifying the therapeutic index of antimetabolites by
administering noncytotoxic compounds dates back more than 30
years.[7,8] The conversion of deoxyuridine monophosphate (dUMP) into
deoxythymidine monophosphate (dTMP) is carried out in two sequential
chemical reactions: First, dTMPS catalyzes the substitution of the
hydrogen in the C-5 position of dUMP with a methylene group; then,
the enzyme reduces the methylene group to a methyl group. The
cofactor that is involved both as the donor of the CH3
group and, subsequently, as the reducing agent is the N5,N10CH2FH4.[9-11]
Therefore, the conversion of uridylate into thymidylate involves the
transient formation of a ternary complex constituted by the substrate
dUMP, the enzyme dTMPS, and the cofactor N5,N10CH2FH4;
with time and in the presence of insufficient concentration of
folate cofactor, the ternary complex dissociates, releasing dTMP, the
free enzyme, and dihydrofolate (FH2), the oxidized
cofactor. After administration of 5-FU, the FdUMP that is generated
will compete with the physiologic substrate dUMP for dTMPS; however,
due to the characteristics of the fluorine-carbon bond, the
substitution of the fluorine atom with the methylene group will not
occur and the reaction does not proceed further.
Activity of dTMPS, size of the cellular pools of dUMP, folate
cofactor, and the amount of FdUMP generated are important
determinants of response to 5-FU in the presence of calcium folinate.
In particular, the presence of suitable amounts of the reduced folate
cofactor in tumor tissues is critical, because a binary complex of
FdUMPdTMPS is relatively weak, whereas the ternary complex of
FdUMP with dTMPS and N5,N10CH2FH4
is more stable and dissociates more slowly.
Key factors associated with the ability to potentiate 5-FU action by
calcium folinate include stabilization of the ternary complex, which
results in a pronounced and prolonged inhibition of the target enzyme
dTMPS and inhibition of DNA synthesis. The duration of dTMPS
inhibition in the presence of calcium folinate is also influenced by
the extent of polyglutamation of N5,N10CH2FH4.
From the data generated to date, the rationale for the combination
of 5-FU with calcium folinate can be summarized as follows:
Calcium folinate potentiates the cytotoxic effects of 5-FU primarily
in tumor cells with relatively low intracellular concentrations of N5,N10CH2FH4,
a folate cofactor essential for the tight binding of FdUMP, the
active metabolite of 5-FU, to the target enzyme dTMPS. Increased
cytotoxicity occurs due to stabilization of the ternary complex of N5,N10CH2FH4dTMPSFdUMP,
which results in prolonged inhibition of dTMPS. Consequently, DNA
synthesis inhibition is more rapid and sustained for a longer duration.
The effect of calcium folinate was dose- and schedule-dependent.
Cytotoxicity was optimal when 20 µmol/L of calcium folinate was
exposed for 24 hours prior to 5-FU administration. Modulation of 5-FU
does occur at lower concentrations, but, when these are used, the
duration of calcium folinate exposure should be longer than 24 hours.
In those tumor cells where incorporation of FUTP into cellular RNA
was the primary site of 5-FU action, calcium folinate shifted the
drug effect to inhibition of deoxyribonucleic acid (DNA) synthesis
via stabilization of the ternary complex.
Polyglutamate forms of N5,N10CH2FH4
are more active potentiators of 5-FU cytotoxicity than
monoglutamates. The extent of polyglutamate formation appears to be a
function of the duration of exposure to calcium folinate.
Schedule of 5-FU Modulation by Calcium Folinate
Table 1 and Table
2 outline the various schedules of 5-FU/calcium folinate
modulation under clinical evaluation, including daily × 5,
weekly × 6 (intravenous push and 24-hour infusion), and
protracted continuous intravenous infusion. The total 5-FU dose
delivered per course of therapy was approximately 1,275 mg/m²
with the daily × 5 schedule; 3,000 mg/m² (intravenous push)
or 15,600 mg/m² (24 hours) for the weekly schedule; and 9,000
mg/m² for protracted infusion. As discussed below, therapeutic
efficacy was achieved with a different profile of toxicity as a
function of the schedule but not the dose of calcium folinate.
Modulators of 5-FU Under Study
Table 3 is a list of the
various 5-FU modulators, which include calcium folinate, modulator of
FdUMP to dTMPS binding (as discussed above); 5-ethynyluracil (776C85)
(irreversible inhibitor of dihydropyrimidine dehydrogenase [DPD], the
enzyme responsible for degradation of 5-FU);
5-chlorodihydropyrimidine (5CDHP), a competitive inhibitor of DPD and
a component of S-1, a new 5-FU prodrug; sodium oxonate, a competitive
inhibitor of 5-phosphoribosyl-1-pyrophosphate transferase, an enzyme
responsible for the metabolism of 5-FU to fluorouridine
monophosphate; and interferon, a known modulator of 5-FU action. A
summary of the site of action of these modulators is presented in Table
Phase III Trials in Colorectal Cancer
Randomized studies of 5-FU vs 5-FU/calcium folinate in patients with
advanced colorectal cancer indicate a response advantage for the
combination over 5-FU alone in six of the seven trials, with reported
response rates of 20% to 30%.
Comparative Toxicity of 5-FU and 5-FU/Calcium Folinate
MyelotoxocityWhere the treatments were designed to be
equitoxic, the 5-FU/calcium folinate arm was generally less
myelotoxic than the 5-FU arm, or myelotoxocity was minor in both
arms. In all of the studies reported, thrombocytopenia was
considerably less frequent than leukopenia.
DiarrheaDiarrhea was a more frequent side effect of
5-FU/calcium folinate than of 5-FU in most studies; it was the major
dose-limiting toxicity of patients treated with the Roswell Park
Cancer Institute weekly schedule and the weekly high-dose FU/calcium
folinate reported by Köhne et al.
StomatitisStomatitis was a prominent toxicity of the
schedules in which 5-FU/calcium folinate was given on a daily ×
5 schedule or in which the calcium folinate was given by continuous
infusion. It was far less important as a toxicity in patients treated
either with the Roswell Park or high-dose 5-FU/calcium folinate schedule.
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