Modulation of 5-Fluorouracil in Mice Using Uridine ......Vol. 3, 309-315. February 1997 Clinical Cancer Research 309 Modulation of 5-Fluorouracil in Mice Using Uridine Diphosphoglucose1

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

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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-



100

0 7 14 21

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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



**

f

4,E

0

0

E

4,

‘5

4)

Maltase

0 7 14 21

6

5

0

0_3

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#{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.


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.




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.



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


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.

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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

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Modulation of 5-Fluorouracil in Mice Using Uridine ......Vol. 3, 309-315. February 1997 Clinical Cancer Research 309 Modulation of 5-Fluorouracil in Mice Using Uridine Diphosphoglucose1