View
3
Download
0
Category
Preview:
Citation preview
Vol. 3, 309-315. February 1997 Clinical Cancer Research 309
Modulation of 5-Fluorouracil in Mice Using Uridine
Diphosphoglucose1
Giovanni Codacci-Pisanelli,2 Judit Kralovanszky,
Clasina L. van der Wilt, Paul Noordhuis,
Joseph R. Coloflore, Daniel S. Martin,
Fabrizio Franchi, and Godefridus J. Peters3Department of Clinical Medicine, University of Rome ‘La Sapienza”.
00185 Roma, Italy 1G. C-P., F. F.]; National Institute of Oncology.
1525 Budapest. Hungary [J. K.]; Memorial Sloan-Kettering CancerCenter. New York. New York 10021 lJ. R. C.. D. S. Mb: andDepartment of Medical Oncology. Free University Hospital. 1007MB, Amsterdam, the Netherlands [C. L. v. d. W., P. N., G. J. P.]
ABSTRACT
Uridine diphosphoglucose (UDPG) is a precursor of
uridine that can be used as a rescuing agent from 5-fluorou-
racil (5FU) toxicity. Four doses of UDPG (2000 mg/kg i.p. or
P.O. at 2, 6, 24, and 30 h after SFU bolus) allowed the
escalation of a weekly bolus of 5FU from 100 mg/kg (5FU1�)
to 150 mg/kg (5FU150) in healthy and tumor-bearing
BALB/c, C57/BI, and CD8F1 (BALB/c X DBA/8) mice.5FU1#{231}0without rescuing agents is not tolerated by the ani-
mals. When followed by UDPG, on the contrary, it is possi-
ble to increase the dose of 5FU even when it is modulated by
beucovorin. Toxicity was the same for SFU1�,�J and 5FU15() +
UDPG, and the nadir values (expressed as a percentage of
pretreatment values) were 83 and 85% for weight, 45 and
45% for hematocrit, and 45 and 61 % for leukocytes, respec-
tively. Platelets were not affected by treatment.
A protective effect was also shown for the gastrointes-tinal tract. The enzymes thymidine kinase, maltase, and
sucrase were measured in the intestinal mucosa at different
times after SFU treatment with or without UDPG rescue.
Even if the nadir values in enzyme activities were similar in
mice receiving or not receiving UDPG, the pattern of recov-
ery showed that cell repopulation was more rapid in the
group treated with UDPG.
5FU150 + UDPG had enhanced antitumor activity
against CD8F1 mammary carcinoma and against the resist-
ant tumor Colon 26 (tumor doubling time 1.9 days forcontrols, 8.5 days for 5FU1($), 13.7 days for 5FU150 + UDPG,
Received 7/I 8/96: revised 1 1/7/96: accepted I I/I 8/96.
The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
I Sponsored by the European Cancer Centre in Amsterdam and in part
by Grant EIT T-03 460/1993 from the Ministry of Welfare of Hungary.
2 Recipient of a fellowship from the European Cancer Centre, Amster-
dam, the Netherlands.
3 To whom requests for reprints should be addressed, at Department ofMedical Oncology. Free University Hospital. P.O. Box 7057, 1007 MB.Amsterdam, the Netherlands. Fax: 31-20-4443844.
and 15.9 days for 5FU150 + leucovorin + UDPG). We
demonstrated that UDPG administered at 2, 24, and 30 h
after 5FU1� does not reduce the antitumor activity of 5FUin two sensitive tumors (Colon 38 and Colon 26-10).
In conclusion, UDPG is a promising rescuing agent for5FU; it reduces the toxic side effects and increases the
therapeutic index.
INTRODUCTION
5FU4 is an essential element in the treatment of several
tumors, and different attempts have been made to improve the
effectiveness of this drug. The complex intracellular metabolism
of SFU makes it a suitable candidate for modulation ( 1 ). En-
counaging results have been obtained with several agents (2),
and the association of5FU with LV is at present the most widely
used treatment for advanced coloreetal cancer.
Similar to what is observed with other anticancer agents, it
is possible to enhance the activity of SFU by increasing the dose.
In the mouse, the administration of high doses of 5FU has been
associated with an increased incorporation of fluoronucleotides
in RNA (3). and a dose-response effect has been demonstrated
in the clinic for SFU alone (4) and for the combination of SFU
with modulating agents (5-7). Dose escalation, however, is
generally prevented by the appearance of toxic side effects that
primarily involve the bone marrow and the GI tract.
Biochemical modulation can be used not only to improve
the antiproliferative effect of SFU but also to reduce the toxicity
of 5FU on normal tissues. Among the different compounds
tested, the most interesting results have been obtained with Urd
(2. 8). Urd rescue from the activity of 5FU has been evaluated
extensively in preclinical systems (9-13). In clinical trials. the
results were encouraging, but toxic side effects of Und appeared
after parentenal and oral administration (14, 15). For this reason,
additional studies have been started to find a more suitable
molecule that is equally effective in rescuing from 5FU toxicity,
but without the side effects of Urd (16, 17).
UDPG is a physiological substance that is normally used in
biochemical reactions as a donor of phosphorylated sugars or as
a precursor of glucuronic acid. It can also be converted into Und,
and previous experiments in mice showed that after administra-
(ion of UDPG. Urd nucleotides increased in the liver but not in
the tumor ( 18). UDPG has been used as a rescuing agent from
the toxic effects of 5FU in mice, and the results were similar to
those obtained with Urd. The MTD of 5FU increased by 50%,
and the antitumor activity of high-dose 5FU improved ( 19).
The aim of the present study was to analyze in detail the
4 The abbreviations used are: 5FU. 5-fluorouracil; UDPG, uridine
diphosphoglucose: Urd, uridine; LV, leucovorin; MTD. maximum tol-
crated dose; TD, doubling time: TK. thymidine kinase; GI, gastrointes-
tinal.
Research. on June 19, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
http://clincancerres.aacrjournals.org/
310 UDPG Modulation of SFU in Mice
protective activity of UDPG from 5FU side effects in terms of
systemic, hematological, and GI toxicity. Furthermore, we
tested the combination of 5FU and UDPG in different tumor
models, both sensitive and resistant to 5FU, to determine the
optimal rescuing schedule to avoid the risk of reducing the
antitumor activity of 5FU.
MATERIALS AND METHODS
Chemicals. SRi was obtained from Hoffman-La Roche
(Mijdrecht, the Netherlands). LV was prepared by the pharmacy
department of the Free University Hospital (Amsterdam, the
Netherlands). UDPG was provided by Boehringer-Mannheim
Italia (Monza, Italy). All drugs were given as i.p. bolus injec-
tions, except for experiments performed on CD8F1 (BALB/c X
DBA/8) mice. All other chemicals are commercially available.
Toxicity Studies. Female BALB/c or C57/Bl mice of
8-10 weeks were obtained from Harlan/Cpb (Zeist, the Neth-
erlands) and maintained in the Clinical Animal Laboratory of
the Free University. All experiments were performed in agree-
ment with the rules for animal welfare established by the local
committee. CD8F1 mice hosting first-generation transplants of
CD8F1 mammary carcinoma were used in a different set of
experiments on oral UDPG.
Systemic toxicity studies were carried out in healthy fe-
male C57IB1 and BALB/c mice. Parameters used to define the
MTD were weight loss not greater than 15% (the initial weight
of the mice was 20-23 g) and/or lethality less than 10%.
SRi was administered at the MTD as defined during the
systemic toxicity studies ( 100 mg/kg for 5FU alone and 150mg/kg when it was followed by UDPG rescue). LV was injected
i.p. in two doses of 50 mg/kg; one dose was given 1 h before
SRi administration and one dose was given together with 5FU
(12). Oral UDPG was given to CD8F1 mice at a dose of 1950mg/kg 2.5 h after SRi administration, then five doses of 2600
mg/kg q 8 h. Parenteral UDPG was initially given at a dose of
2000 mg/kg, repeated four times at 2, 6, 24, and 30 h after SRI
administration. In a later phase of the study, the injection at 6 h
was omitted.
Investigations on hematological toxicity were performed as
described previously (12). Blood was drawn from the retro-
orbital plexus with heparmnized capillaries after slight ether
anesthesia. Initial values (mean ± SD for five animals) for
hematological parameters were: hematocrit, 42 ± 1 .2%; leuko-
cytes, 4700 ± 600 cells/mb; and platelets, 4.5 ± 0.6 X 100,000
cells/mb.
GI toxicity was studied as described previously (20). SRI
was administered once as an i.p. bolus at a dose of 200 mg/kg.
Briefly, mice treated with SFU with or without UDPG rescue
were sacrificed at different time points. The small intestine was
removed, washed with normal saline containing I mM DTI’,
and frozen in liquid nitrogen. After homogenization, TK activity
was determined by measuring the conversion of 2-[’4C]thymi-
dine to 2-[t4C}thymidine-monophosphate; sucrase and maltase
activity and protein content were determined according to pub-
bished methods (20).
Antitumor Activity. Antitumor activity was studied onthe murine colon carcinomas Colon 26 and Colon 26- 10 main-
tamed in female BALB/c mice, Colon 38 maintained in CS7IB1
mice as described previously ( 1 2), and on mammary tumor
CD8F1 (18). Tumors were transplanted s.c. in the thoraeic
region on each flank of the animals. Tumor size was measured
sequentially twice weekly using a caliper. and the volume was
calculated as length X width X height X 0.5. The evaluation of
antitumor activity was based on the ratio of the mean volume of
treated tumors and the mean volume of control tumors. The
growth delay factor was calculated from the median tumor TD
of the tumors of treated mice and control mice according to the
following formula: growth delay factor = (TDtreated TD�0�
trois)/TDcontrois. For Colon 26, TDcontrots �5 about 3 days,
whereas it is 4.5 days for Colon 26-10 and 5 days for Colon 38
( 1 1 ). Treatment was started when the tumors had reached a sizeofSO-lSO mm3 (approximately 10 days for Colon 26 and Colon
26-10 and 18 days for Colon 38). For CD8F1 tumors, only the
final weight of the tumor (measured 6 days after the third course
of treatment) was considered.
Analysis of Plasma Concentrations of UDPG and Urd.
Plasma was obtained from heparinized blood drawn from the
retro-orbital plexus. Plasma was deproteinized by addition of
cold trichboroacetie acid to a final concentration of 8% and by
keeping it on ice for 15 mm. After centrifugation (5000 X g for
15 mm), the supernatant was neutralized by mixing with two
volumes of trioctylamine-freon (1 :4) and centrifuged, and the
aqueous part was recovered and stored at - 20#{176}C(2 1).
High-performance liquid chromatography analysis was
performed isocratically on a C18-p.Bondapaek column with 50
mmol/b KHIPO4, 5 mmol/l tetrabutyl ammonium hydrogen sub-
phate, and 2% methanol (v/v, pH 3.5), flow rate 1 mI/mm. Peaks
were detected by UV analysis at 254 and 280 nm. Retention
times were 3.70 mm for uracil, 4.60 mm for Urd, and 13.60 mm
for UDPG. Concentrations were calculated from the integrated
peaks using an AXXIOM system (Separations, the Nether-
lands).
RESULTS
UDPG Schedule. UDPG can be administered accordingto several schedules, p.o. or i.p. We tested different rescuing
regimens to identify the optimal one. In the first set of experi-
ments, UDPG was given p.o. over 2 days as described in
“Materials and Methods,” then we used the parenteral adminis-
tration of UDPG at similar time points (2, 6, 24, and 30 h after
5FU injection). In the course of our research, however, it be-
came clear that it was possible to eliminate the dose at 6 h with
no reduction in the rescuing effect of the treatment. Because this
protocol was also more similar to that already used with Urd (9,
1 1), only data from BALB/e and C57/Bl mice that received this
rescuing regimen are reported in this paper. CD8F1 mice re-
ceived the oral rescue regimen.
Systemic Toxicity. The toxicity of different doses of
SFU, with or without UDPG rescue, was studied in healthy
BALB/e mice. The results for the systemic toxicity (weight loss)
are shown in Fig. 1 . The MTD of SFU with no rescue, given as
a weekly bolus repeated two or three times, was 100 mg/kg in
both BALB/e and CS7IBI mice. This dose was well tolerated by
the animals, and it caused a maximum weight loss of - 15%. A
dose of 150 mg/kg was lethal by day 12, but it could be safely
administered when it was followed by UDPG rescue. The max-
Research. on June 19, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
http://clincancerres.aacrjournals.org/
100
0 7 14 21
C
tS>
tS
C
0
4)
‘5
‘5
C
0
7 14 21
0 7 14 21
hematocrit
0 7 14
Days after first treatment
21
Clinical Cancer Research 311
.�
IC
,� 80
1’ 1’ .Days after first treatment
Fig. 1 Systemic toxicity of 5FU ± UDPG. Relative weight (comparedto that before treatment) of healthy BALB/c mice treated with weekly
5F1�JRx) (-A-), SFUISO (-#{149}-). or 5-FUIS() + UDPG (-LI-). Data ( ±SE) are
given for groups of three to five mice. Treatment is indicated by the
arrotvs.
imum weight loss was -10% at day 19. The highest 5FU dose
tested (200 mg/kg) was too toxic even when combined with
UDPG.
A dose of 150 mg/kg 5FU was considered the MTD for the
combination, and 5FUIS() + UDPG was used in the studies on
hematological toxicity and on antitumor activity. In mice, the
systemic toxicity of 5FU was not enhanced by the addition of
LV, and the same dose of 5FU could also be used when
modulation with LV was used (12).
Hematological Toxicity. These studies were performed
in healthy BALB/c mice by comparison of the standard MTD of
5FU (100 mg/kg) with 5FUtS() + UDPG. Results expressed as
a percentage of the initial value are shown in Fig. 2. 5FU
administration caused a sharp decrease in hematoenit, with a
nadir value of42.8 at day 21 for 5FUtOO and of58.3 at day 15
for SFUIS() + UDPG. Changes in WBCs were quite similar,
with nadirs of 49.3 and 62.7, respectively, at day 16. In both
eases, an overshoot was observed after the end of treatment.
Platelets were not reduced in either case, but a striking rebound
was observed 2 weeks after the first dose for both protocols.
GI Toxicity. The effect of UDPG on the GI toxicity of5FU was evaluated by measuring the protein content of the
intestinal wall and the activity of enzymatic markers of cell
proliferation (TK) and of enterocytie differentiation (suerase
and maltase) in the mucosa.
The mucosa of the small intestine was damaged to a similar
extent by 5FU treatment, independent of UDPG administration,
but the rescuing protocol allowed a faster recovery of cell
functions. The decrease in TK activity is very fast (6-24 h) and
is followed by a rebound, whereas digestive enzymes (maltase
and sucrase) decrease more slowly and reach a nadir only on day
3. This suggests that their change depends on the antiprolifena-
tive effect of SFU that inhibits the replacement of functionally
mature enterocytes. Results are shown in Fig. 3.
Cell proliferation was estimated by measuring TK, and this
parameter gave the best indication of the protective activity of
UDPG. After a short-lasting reduction in the proliferative activ-
‘5
‘5
C
0
Fig. 2 Hematological toxicity of 5FUI�X� (-Li-) compared with 5FU15()+ UDPG (-LI-). Data (±SE) are given for groups of three to five miceand are expressed as percentage of initial value. On day 28. all values
were within the range of controls. Statistical analysis: hematocnit values
of treated mice are different from control animals (-#{149}-) at all times fromday 6 on (P < 0.05). Leukocyte and platelet counts are different from
controls as indicated (*, P < 0.()05; **, P < 0.001).
ity, the mueosa of mice receiving UDPG was actively regener-
ating already on day 4 (P < 0.0 1 both versus controls and versus
animals not treated with UDPG). On day 7, TK activity in mice
treated with UDPG was within normal ranges, whereas it was
still high in mice that were not rescued (P < 0.01 both versus
controls and versus animals treated with UDPG).
The protein content was initially slightly increased, then it
was significantly reduced in both treatment groups. In animals
receiving UDPG, however, protein content was normal on day
4, when in the other group it was still significantly lower than
that of the controls.
The time course of the markers of enterocytie differentia-
tion was similar to that of the protein content. Sucrase was lower
Research. on June 19, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
http://clincancerres.aacrjournals.org/
**
f
4,E
0
0
E
4,
‘5
4)
Maltase
0 7 14 21
6
5
0
0_3
E
#{163}
0
C
0
30
0
a.
4)
E1.)
a’E
**
I ** �
0
Protein
312 UDPG Modulation of SRi in Mice
Thymidine kinase
Sucrase
0 2 4 6 8 10
days after 5FU treatment
Fig. 3 Protective effect of UDPG on the GI mucosa: controls (-#{149}-);
�‘�2OO () and 5FU2m + UDPG (-U-). Data are given as mean(±SE) for five animals. Different from controls (*, P < 0.05; **, P <0.01). Different from 5FU without rescue (##, P < 0.01).
than controls on day 2, but in the group of mice treated with
UDPG, it was within the normal range already on day 3, when
it was still low in the other group. Mabtase was lower than
controls on day 7 in the group not receiving UDPG.
Days after first treatment
Fig. 4 Effect of treatment with 5FUI()) (-A-), 5FU1,() + UDPG (-LI-),and 5FUISO -LV + UDPG (�-) on the resistant tumor Colon 26.Untreated controls, (-#{149}-). Tumor volume is expressed as percentage ofthe initial value for each tumor (mean ± SE). Each group consisted of10-12 tumors at the beginning of the experiment. *. different from the
group treated with 5-FU1()) (P < 0.05); #. different from the grouptreated with 5-FUIS() + UDPG (P 0.028).
Antitumor Activity. The therapeutic efficacy of the dif-
ferent doses of SFU, followed by UDPG rescue when required,
was studied in three sensitive tumors (Colon 26-10, Colon 38,
and CD8F1 ) and in the resistant tumor Colon 26.
In Colon 26, maintained in BALB/c mice, we studied the
ability of SFUIS() + UDPG to give a better therapeutic effect
compared to 5FUt(x) (Fig. 4). The untreated controls presented
rapid growth, the tumors caused eachexia, and the survival was
very short. 5FU100 reduced the growth rate only for a limited
time. The higher dose of 5FU gave better results. and these were
further improved by combination with LV (see Table 1 ). These
data prove that high-dose SFU with UDPG rescue actually
presents a better antitumor activity. These results were con-
firmed in Colon 26-10 and in CD8F1; tumor growth was more
effectively inhibited by high-dose 5FU with UDPG rescue com-
pared to standard treatment.
To exclude the possibility that UDPG rescue might be
interfering with the antitumor activity of SFU, we compared
5FU at the standard dose ( 100 mg/kg) with or without UDPG in
the sensitive tumors Colon 26-10 and Colon 38. Colon 38
tumors in mice treated with 5FU1�� and those of mice treated
with 5FU)(5) + UDPG do not show any significant difference in
growth rate (Fig. 5) non in the parameters used for the evaluation
of the treatment effects (see Table I ). However, some prelimi-
nary experiments in Colon 26-10 and Colon 38 showed that in
Colon 26-10, it was indeed possible to reduce the activity of
5FU when an additional injection of UDPG was given after 6 h.
However, this did not occur with Colon 38. We tested different
schedules and determined that by skipping the dose of UDPG
after 6 h, there was no risk of tumor protection.
Analysis of Plasma Concentrations. We measured the
plasma concentration of UDPG and of Urd at several time points
after the administration of a bolus of UDPG at a dose of 2000
mg/kg (Fig. 6). Peak plasma concentrations were 424 ± 161
mM at 10 mm for UDPG and 164 ± 13 mM at 20 mm for Urd.
Research. on June 19, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
http://clincancerres.aacrjournals.org/
Clinical Cancer Research 313
Table I Antitu mor activity o f 5FU in different do ses ± UDPG rescue
Minimum Tumor
GDF” T/C5 Tumor TD’ Survival” weighte weight’
Colon 26Controls 1.9 245-FUt(s) 3.4 42 8.6 35 84.3 ± 12.9 286.7
5-FU,50 + UDPG 6.1 47 13.8 35 92.3 ± 5.1 218.45-FU15() + UDPG + LV 7.2 38 15.9 31 89.3 ± 11.2 151
Colon 26-10Controls 3.8 23’5-FU,0() NR 28 NR 32’ 85.5 ± 8.8 272
5-FUt�,) + UDPG 2.2 16.5 12.1 32’ 97.2 ± 3.2 290
Colon 38Controls 3.3 46’ >10005-FUI(x) 7.7 7.8 28.7 61’ 94.9 ± 2.2 42.85-FU1,,�) + UDPG 6 2.8 27 61’ 87.7 ± 7.8 65.2
CD8F1
Controls 31815’F’��ttX) 6885-FUt,) + UDPG” 212
�1 GDF. growth delay factor. NR, not reached.S The mean volume of the tumors of treated mice divided by the values obtained for control animals (%). The maximal TIC is given: for Colon
26 tumors, calculation is limited to the period that control mice can be kept in experiment (see also Fig. 4).
(. Tumor ID is calculated from the first day of treatment.d Survival is calculated from the day of tumor transplantation.
e The minimum weight (expressed as percentage of initial weight) observed in the 2 weeks from the first treatment.
I Weight in milligrams 6 days after the third course of treatment. For Colon 26. Colon 26- 10. and Colon 38, the weight was estimated from the
volume (assuming 1000 mm3 1 g); for some tumors, the weight of controls is not given due to short survival of controls (Colon 26) or to sacrificeof the mice when tumor volume exceeded 1000 mm3.
S Mice were sacrificed when tumor size was > 1000 mm3. Mice bearing Colon 26 were sacrificed I day before suspected death due to cachexia.h UDPG given orally (see Materials and Methods”).
After 60 mm, Und was -50 mM, whereas the parent compound
was no more detectable.
DISCUSSION
UDPG rescue enhanced the therapeutic efficacy of 5FU in
resistant tumors because a higher dose of 5FU can be adminis-
tered to mice. UDPG reduced the systemic and hematological
toxicity of this drug. In 5FU-sensitive tumors. the antitumor
activity was not affected, even when the standard dose of SFU
was followed by rescue. The better therapeutic effect of high-
dose 5FU with UDPG rescue could be further improved by
combination with LV.
Previous experiments on the modulation of 5FU or 5’-
deoxy-5-fluorouridine with the simultaneous administration of
nueleosides not only increased the activity but also the toxicity
of the treatment (22, 23). On the other hand, it was possible to
increase the dose of 5FU when a rescuing protocol was used (9,
I I , 24). The administration of an excess of Urd shortly after
5FU theoretically might reduce the antitumor activity (25), but
a protective effect of UDPG on the tumor was specifically
excluded.
The final dose and the schedule selected for UDPG rescue
were based on previous data ( 19) and on the experience with the
P.O., � and iv. administration of Urd ( I 1 ). UDPG was used
more efficiently than Urd because the dose of UDPG was lower
than that of Und, especially when calculated on a molar basis:
2000 versus 3500 mg/kg and 3.2 versus 14.3 mmollkg, with
cumulative doses of 9.6 mmol for UDPG and 28.6 mmol for
Urd. In previous studies, it was observed that high-dose Urd
would result in plasma levels of -20 mM (26), but rescue was
also possible at lower concentrations of Urd (- 100 p.M; Refs.
8, 1 1 , and 27) when maintained for a longer period. Also, in
some initial experiments on Urd rescue, the prolonged admin-
istration of low doses proved to be as efficient as bolus injec-
(ions ofhigh doses (9, 10). Although the measurement of plasma
Urd levels is indicative of the tissue distribution, concentrative
mechanisms for Urd are present and may alter its concentration
in tissues (28). We therefore selected a lower dose for each
rescuing injection but repeated it until 30 h after 5FU adminis-
tration.
Similar to Urd, several protocols of UDPG administration
could rescue mice from SFU-induced toxicity, which allowed a
50% increase in the MTD ( 19). The increased therapeutic indexof high-dose SRI with UDPG (present study) is in agreement
with clinical evidence of a dose-response relationship for anti-
tumor activity for both 5FU alone (4) and 5FU modulated with
LV (6, 7). The possibility of increasing the dose of SFU when
followed by Urd rescue in patients has also been demonstrated
(15, 25, 27, 29). Unfortunately, the clinical use of Urd is
hampered by the presence of specific toxic side effects, phlebitis
and fever, after continuous infusions, whereas oral administra-
tion caused diarrhea (8). Fever was also observed in rabbits (30,
3 1 ), whereas in mice, high-dose Urd caused hypothermia (21).
This effect could be partially prevented by inhibition of Urd-
phosphorylase with benzylaeyclouridine (21). No evidence of
alterations of body temperature was seen in mice throughout the
experiments described in the present paper with UDPG used at
the indicated dose.
Research. on June 19, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
http://clincancerres.aacrjournals.org/
400
100
10
0E
C0‘5L.
C
0C0
0
0 1 10 15 20 30 60 120180240time (minutes)
Fig. 6 Plasma concentrations of UDPG (-0-) and Urd (-U-) afterinjection of 1500 mg/kg UDPG. Values are in p.mol/l and are means ±SE of four to eight mice.1000
100
0
E
0
0
E0
‘5
0
314 UDPG Modulation of SRi in Mice
:
E
0
0
E
C
‘5
0 100
60
10
0 7 14 21 28 35
Colon 38T T
�.T/
/\�
0 7 14 21 28 35
Days after first treatment
Fig. 5 Effect oftreatment with 5-FU� (-L:�-) versus 5-FU� + UDPG(�A�) on the SRi-sensitive tumors Colon 26-10 and Colon 38. Untreated
controls (-#{149}-). Tumor volume is expressed as percentage of the initialvalue for each tumor (mean ± SE). For Colon 26- 10, values for the twotreatments are never statistically different; for Colon 38 they are onlydifferent on day 8 (P = 0.017).
An increase in antitumor activity was obtained not only in
sensitive tumor lines (Colon 26-10 and Colon 38) but, more
importantly, also in the resistant tumor Colon 26. This is in
contrast to results obtained with LV modulation in which, in
vitro, an enhancing effect could only be obtained on lines
“intrinsically sensitive to SFU” (32). It is also important to note
that nucleotide metabolism varies in the different tissues and
may specifically affect the rescuing activity of Urd on normal
tissues and not on tumors. Colon 38, for example, has been
reported to possess a limited concentrating capacity for Urd (26,
28, 33); therefore, it was very important to verify that our
rescuing protocol did not affect the antitumor activity of SFU in
this tumor.
In a previous study, it was demonstrated that Urd could
reduce the toxicity of the standard dose of SRI (1 1), but this is
the first description of the hematobogical toxicity of �
compared to SFU1#{216}#{216}.The pattern of toxicity of S�FUim is
similar to that of S-FU150 with UDPG, but the recovery tended
to be more rapid with a rescue protocol. Contrary to what is
commonly observed in clinical practice, in mice SRI caused a
significant decrease in hematocrit (1 1) but did not cause a
substantial decrease in platelets. Also, in the clinical application,
Urd rescue was successful in reducing leucopenia, but throm-
bocytopenia could not be reduced (15).
0! toxicity remains an important drawback to the use of
high-dose SRI; therefore, we studied the effect of UDPG rescue
on the damage caused by SRI on the intestinal mucosa of mice.
The dose of SRI was slightly higher in this set of experiments
(200 mg/kg) and has been selected according to previous results
as a dose that would induce significant changes in all aspects of
mucosab proliferation and function (20). Although the combina-
(ion of SFU with the rescuing agent did not prevent GI toxicity,
the quick recovery of cell proliferation (already evident at 72 h)
resulted in a faster normalization of intestinal functions as
indicated by the recovery in maltase and suerase activity. The
protective effect on the GI mucosa is particularly relevant for
the clinical use of 5FU modulated with LV. The combination of
myelosuppression and extensive mucosal damage was in fact
responsible for several episodes of life-threatening toxicity ob-
served in the early use of this combination (34).
UDPG is an effective rescuing agent from the toxicity of
SRI, allowing a 50% increase in dose intensity. It did not
interfere with the antitumor activity of SFU and did not cause
any of the toxic side effects of Urd. Its clinical use in combi-
nation with high-dose SRI may improve the activity of SRI as
a single agent and of the combination SFU-LV that represents
the most widely used treatment for advanced coboreetal carci-
noma.
REFERENCES
I . Pinedo, H. M., and Peters, G. J. Fluorouracib: biochemistry andpharmacology. J. Clin. Oncol., 6: 1653-1664, 1988.
2. Peters, G. J., and Van Groeningen, C. J. Clinical relevance ofbiochemical modulation of 5-fluorouracil. Ann. Oncol.. 2.’ 469-480,1991.
3. Nord, L. D., Stobfi, R. L., and Martin, D. S. Biochemical modulation
of 5-fluorouracil with leucovorin or delayed uridine rescue. Biochem.Pharmacol., 43: 2543-2549, 1992.
4. Hryniuk, W. M. The importance of dose-intensity in the outcome of
chemotherapy. In: V. I. Dc Vita, S. Hellman, and S. A. Rosenberg(edt.), Important Advances in Oncology, pp. 121-141. Philadelphia:J. B. Lippineott Co., 1988.
5. Ardaban, B., Singh, G., and Silberman, H. A randomized Phase I andII study of short-term infusion of high-dose fluorouracib with or without
Research. on June 19, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
http://clincancerres.aacrjournals.org/
Clinical Cancer Research 315
N-(phosphonacetyl)-L-aspartic acid in patients with advanced pancreatic
and colorectal carcinoma. J. Clin. Oncol., 6: 1053-1058, 1988.
6. Arbuck, S. G. Overview of clinical trials using 5-fluorouracil andleucovorin for the treatment of colorectal cancer. Cancer (Phila.), 63:1036-1044, 1989.
7. Brohee, D. 5-fluorouracil and folinic acid in the treatment of ad-vanced colorectal cancer: an updated meta-analysis. Med. Sci. Res., 22:
383-384, 1994.
8. Van Groeningen, C. J., Peters, G. J., and Pinedo, H. M. Modulation
offluorouracil toxicity with uridine. Semin. Oncol., 19: 148-154, 1992.
9. Martin, D. S.. Stolfi, R. L., Sawyer, R. C., Spiegelman, S., and
Young, C. W. High-dose 5-fluorouracil with delayed uridine “rescue” in
mice. Cancer Res., 42: 3964-3970, 1982.
10. Klubes, P., and Cerna, I. Use of uridine rescue to enhance theantitumor selectivity of 5-fluorouracil. Cancer Res., 43: 3182-3186.1983.
11. Peters, G. J., Van Dijk, J., Laurensse, E., Van Groeningen, C. J.,
Lankelma, J., Leyva, A., Nadal, J. C., and Pinedo, H. M. In vitrobiochemical and in vivo biological studies of the uridine rescue of
5-fluorouracil. Br. J. Cancer, 57: 259-265, 1988.
12. Nadal, J. C., Van Groeningen, C. J., Pinedo, H. M., and Peters, G. J.
Schedule-dependency of in vivo modulation of 5-fluorouracil by leu-covorin and uridine in murine colon carcinoma. Invest. New Drugs, 7:163-172, 1989.
13. Kiubes, P., and Leyland-Jones, B. Enhancement of the antitumor
activity of 5-fluorouracil by uridine rescue. Pharmacol. Ther., 41: 289-
303, 1989.
14. Van Groeningen, C. J., Leyva, A., Kraal, I., Peters, G. J., andPinedo, H. M. Clinical and pharmacokinetic study of prolonged admin-istration of high-dose uridine intended for rescue from 5-FU toxicity.
Cancer Treat. Rep., 70: 745-750, 1986.
15. Van Groeningen, C. J., Peters, G. J., Leyva, A., Laurensse, E., andPinedo, H. M. Reversal of 5-fluorouracil-induced myelosuppression byprolonged administration of high-dose uridine. J. Nail. Cancer Inst., 81:157-162, 1989.
16. Tognella, S. Uridine rescue of the FUra toxicity. The possible roleof uridine diphosphoglucose (UDPG). Anticancer Res., 12: 1923-1924,
1992.
17. Conti, J. A., Kemeny, N., Kelly, J., Gonzalez, G., Bertino, J., and
Martin, D. S. Triacetyluridine (TAU) as rescue from 5-fluorouracil (FU)toxicity allows increased FU dose intensity when given with N-phos-
phonacetyl-L-aspartate (PALA) and methotrexate in advanced colorectal
carcinoma. Proc. Am. Soc. Clin. Oncol., 14: 485, 1995.
18. Colofiore, J. R., Sawyer, R. C., Balis, M. E., and Martin, D. S.Effect of uridine diphosphoglucose on levels of 5-phosphoribosyl py-rophosphate and uridine triphosphate in murine tissues. Pharm. Res., 6:863-866, 1989.
19. Colofiore, J. R., StoWz, R. L., and Martin, D. S. Uridine diphospho-glucose (UDPG) as an alternative to free uridine (UR) for the rescuefrom 5-fluorouracil (FU)-associated toxicity. Proc. Am. Assoc. Cancer
Res., 32: 340, 1991.
20. Bagrij. T., Kralovanszky, J., Gyergyay, F., Kiss, E., and Peters,
G. J. Influence of undine treatment in mice on the protection of
gastrointestinal toxicity caused by 5-fluorouracil. Anticancer Res., /3.
789-794, 1993.
21. Peters, G. J., Van Groeningen, C. J., Laurensse, E., Lankelma, J.,
Leyva, A., and Pinedo, H. M. Uridine-induced hypothermia in mice andrats in relation to plasma and tissue levels of uridine and its metabolites.
Cancer Chemother. Pharmacol., 20: 101-108, 1987.
22. Hartman, H. R., and Bollag, W. Modulation of the effect of fluoro-
pyrimidines on toxicity and tumor inhibition in rodents by uridine and
thymidine. Med. Oncol. Tumor Pharmacother., 3: 1 1 1-1 18, 1986.
23. Au, J. L. S., Wientjes, M. G., and Bramer, S. L. Effect of uridinecoadministration on 5’-deoxy-5-fluorouridine disposition in rats. Cancer
Chemother. Pharmacol., 22: 5-10, 1988.
24. Klubes, P., Cerna, I., and Meldon, M. A. Uridine rescue from the
lethal toxicity of 5-fluorouracil in mice. Cancer Chemother. Pharmacol.,8: 17-21, 1982.
25. Christman, K., Schwartz, G., Saltz, L., Dougherty, J., Casper, E.,Yao, T., Toomasi, F.. Friedrich, C., Martin, D., Bertino, J., and Kelsen,D. Uridine (Urd) allows dose-intensification of FAMTX (5-fluorouracil(FU), Adriamycin (A), Methotrexate (Mtx)). Proc. Am. Soc. Clin.Oncol., 12: 200, 1993.
26. Peters, G. J., Laurensse, E., Leyva, A., and Pinedo, H. M. Purinenucleosides as cell-specific modulators of 5-fluorouracil metabolism
and cytotoxicity. Eur. J. Cancer & Clin. Oncol., 12: 1869-1881, 1987.
27. Van Groeningen, C. J., Peters, G. J., and Pinedo, H. M. Reversal of5-fluorouracil-induced toxicity by oral administration of uridine. Ann.
Oncol.. 4: 317-320, 1993.
28. Damowski. J. W., and Handschumacher, R. E. Tissue uridine pools:
evidence in vito of a concentrative mechanism for uridine uptake.Cancer Res., 46: 3490-3494, 1986.
29. Seiter, K., Kemeny, N., Martin, D., Schneider, A., Williams, L.,
Colofiore, J., and Sawyer, R. Uridine allows dose escalation of 5-flu-orouracil when given with N-phosphonacetyl-L-aspartate, methotrexate,
and leucovorin. Cancer (Phila.), 71: 1875-1881, 1993.
30. Cradock, J. C., Vishnuvajjala, B. R., Chin, T. F., Hochstein, H. D.,and Ackerman, S. K. Uridine-induced hyperthermia in the rabbit.J. Pharm. Pharmacol., 38: 226-229, 1986.
3 1 . Peters, G. J., Van Groeningen, C. J., Laurensse, E., Kraal, I., Leyva,
A., Lankelma, J., and Pinedo, H. M. Effect of pyrimidine nucleosides onbody temperatures of man and rabbit in relation to pharmacokineticdata. Pharm. Res., 4: 113-119, 1987.
32. Beck, A., Etienne, M. C., Cheradame, S., Fischel, J. L., Formento,P., Guillot, T.. and Milano, G. Wide range for optimal concentration offolinic acid in fluorouracil modulation: experimental data on human
tumour cell lines. Eur. J. Cancer, 30A: 1522-1526, 1994.
33. Peters, G. J., Van der Wilt, C. L.. Smid, K., Ruiz van Haperen. V.,Veerman, G.. Laurensse, E., and Pinedo, H. M. Pyrimidine metabolismin murine colon cancer models: relevance for chemotherapy with anti-
metabolites. Biochem. Pharmacol. (Life Sci. Adv.), 9: 365-373, 1990.
34. Grem, J. L., Shoemaker, D. D., Petrelli, N. J., and Douglass, H. 0.Severe life-threatening toxicities observed in study using leucovorin
with 5-fluorouracil. J. Clin. Oncol., 5: 1704, 1987.
Research. on June 19, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
http://clincancerres.aacrjournals.org/
1997;3:309-315. Clin Cancer Res G Codacci-Pisanelli, J Kralovanszky, C L van der Wilt, et al. diphosphoglucose.Modulation of 5-fluorouracil in mice using uridine
Updated version
http://clincancerres.aacrjournals.org/content/3/2/309
Access the most recent version of this article at:
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
.pubs@aacr.orgDepartment at
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://clincancerres.aacrjournals.org/content/3/2/309To request permission to re-use all or part of this article, use this link
Research. on June 19, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
http://clincancerres.aacrjournals.org/content/3/2/309http://clincancerres.aacrjournals.org/cgi/alertsmailto:pubs@aacr.orghttp://clincancerres.aacrjournals.org/content/3/2/309http://clincancerres.aacrjournals.org/
Recommended