effect of (e)-5-(2-bromovinyl)uracil on the …...effect of (e)-5-(2-bromovinyl)uracil on the...

[CANCER RESEARCH 46, 1094-1101, March 1986]

Effect of (E)-5-(2-Bromovinyl)uracil on the Catabolism and Antitumor Activity of5-Fluorouracil in Rats and Leukemic Mice1

Claude Desgranges,2 Gabriel Razaka, Erik De Clercq, Piet Herdewijn,3 Jan Balzarmi, F. Drouillet,

and Henri Bricaud

UnitÃ©8 de l'Institut National de la SantÃ©et de la Recherche MÃ©dicale,Avenue du Haut-LÃ©vÃªque,33600 Ressac, France [C. D., G. R., F. D., H. B.Â¡,and Rega Institute for

MÃ©dicalResearch, Katholieke Universiteit Leuven, Minderbroedersstraat 10, 6-3000 Leuven, Belgium [C. D., E. D. C., P. H., J. B.]

ABSTRACT

In contrast to thymine and 5-fluorouracil (FUra) which werecleared from the bloodstream within 2-4 h after their i.p. administration (200 Â¿imol/kg)to rats, (Â£)-5-(2-bromovinyl)uracil (BVUra)maintained a concentration of 50-70 U.Mfor at least 6 h and was

still present in the plasma 24 h after its administration. In vitroexperiments with rat liver extracts indicated that BVUra was nota substrate but an inhibitor for the reductive step in pyrimidinedegradation catalyzed by dihydrothymine dehydrogenase. Kinetic and dialysis experiments suggested that BVUra was anirreversible inhibitor of this enzyme. The binding of BVUra to theenzyme depended on the presence of reduced nicotinamideadenine dinucleotide phosphate in the reaction mixture. Dihydrothymine dehydrogenase activity was also inhibited in the dialysed105,000 x g supernatant fraction of livers from rats that hadpreviously been treated with BVUra. Such inhibitory effects alsooccurred in vivo; previous adminstration of BVUra increased theplasma half-lives of thymine and FUra by 10- and 5-fold and theirarea under the curve by 9- and 8-fold, respectively.

The effect of BVUra on the antitumor activity of FUra wasevaluated in DBA/2 mice inoculated with 106 P388 leukemia

cells. The mean survival times for the control and FUra-treated

mice (5 mg/kg at 1, 3, 5, and 7 days after tumor cell inoculation)were 9.7 and 12.4 days, respectively. When BVUra (200 /Â¿mol/kg) was administered 1 h before each injection of FUra, the meansurvival time was extended to 17.1 days. BVUra alone did notaffect the mean survival time. When the dose of FUra wasincreased to 20 mg/kg, the mean survival time was 15.3 days;upon a preceding injection of BVUra the mean survival timedecreased to 9.2 days. The latter effect probably resulted froman increased toxicity of FUra. Similar results were obtained ifFUra was replaced by 5-fluoro-2'-deoxyuridine and BVUra by(Â£)-5-(2-bromovinyl)-2'-deoxyuridine. The enhancement of both

the antitumor and toxic effects of FUra by BVUra were mostprobably due to an inhibition of FUra degradation, since, like inrats, BVUra increased the plasma half-life of FUra in DBA/2 mice.

Hence BVUra appears to be an interesting compound, increasingthe potency of FUra by decreasing its degradation.

Received 7/20/84; revised 10/28/85; accepted 11/22/85.' This investigation was supported by grants from the Institut National de la

SantÃ©et de la Recherche MÃ©dicale,the University of Bordeaux II. the BelgianFonds voor Geneeskundig Wetenschappelijk Onderzoek, and the Belgian Gecon-

certeerde Onderzoeksacties.2 Recipient of a fellowship award from North Atlantic Treaty Organization. To

whom requests for reprints should be addressed.3Senior Research Assistant of the Belgian National Fonds voor Wetenschap

pelijk Onderzoek.

INTRODUCTION

Thy4 and Ura are both subject to the same catabolic pathway

which is, in contrast to that of the purine bases, a reductivepathway (see Ref. 1 for review). The first step of the catabolismof Thy and Ura is the reduction of the 5:6 double bond of thepyrimidine ring, followed by the cleavage of the 3:4 bond to give0-ureidoisobutyric acid and /3-ureidopropionic acid from which ÃŸ-

amino acids are formed with the release of ammonia and carbondioxide (2-5). The first enzyme of this catabolic pathway, H2Thydehydrogenase (EC 1.3.1.2), was identified as the rate-limitingenzyme (5-8). The liver appears to be the major site of pyrimidinecatabolism (7-9); the activity of H2Thy dehydrogenase in the liveris 20-fold higher than in lung, bone marrow, and colon (10).

5-Substituted Ura analogues and in particular 5-halogenated

Ura are also substrates for this enzyme (11,12), and it is obviousthat the degradation of FUra via H2Thy dehydrogenase mayaffect the therapeutic efficacy of FUra in the treatment of cancer.The use of H2Thy dehydrogenase inhibitors may be helpful notonly for the elucidation of the mechanism of action of FUra andits analogues but also with respect to an enhancement of theirtherapeutic activity. Some inhibitors have been described previously; among the Ura analogues (9, 13-15), 5-cyanouracil (11,16, 17) and DUra (10, 15, 16, 18-20) are the most potent

inhibitors of pyrimidine degradation in vitro or in vivo. Most ofthese compounds act in a competitive manner, since they arealso substrates of H2Thy dehydrogenase (11-14). Some of them

act in vivo, both by decreasing the degradation of Thy and FUraand by increasing their incorporation into DNA and RNA (18,19).As a consequence, they not only enhance the antitumoral activityof FUra but also its toxicity (17,18). While we were studying thecatabolism of the potent and selective antiherpes agent, BVdUrd(21) in the rat, we found that its degradation product BVUra (Fig.1), in contrast to other pyrimidine bases, had a relatively longhalf-life in plasma (22). We have now pursued these observa

tions, and here we demonstrate that BVUra, being itself notactive as a substrate, reduces the degradation of other pyrimidine bases, such as FUra, by inhibition of the H2Thy dehydrogenase activity in vivo and in vitro. Moreover, BVUra enhancesthe antitumor activity of FUra in the P388 leukemia model inDBA/2 mice.

4The abbreviations used are: Thy, thymine; Ura, uracil; H2Thy, dihydrothymine;FUra, 5-fluorouracil; DUra, 5-diazouracil; BVdUrd, (Â£)-5-(2-bromovinyl)-2'-deoxyu-ridine; BVUra, (E)-5-(2-bromovinyl)uracil; EtUra, 5-ethyluracil; 6-AThy, 6-aminothy-mine; AUC, area under the curve; FdUrd, 5-fluoro-2'-deoxyuridine; dThd, thymidine;FUMP, 5-fluorouridine-5'-monophosphate; HPLC, high-performance liquid chro-

matography.

CANCER RESEARCH VOL. 46 MARCH 1986

1094

Research. on September 2, 2020. © 1986 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

INHIBITION OF PYRIMIDINE DEGRADATION BY BROMOVINYLURACIL

MATERIALS AND METHODS

Chemicals. Thy, FUra, DUra, other Ura analogues, their correspondingdeoxynucleosides and NADPH were obtained from Sigma Chemical Co.,St. Louis, MO. EtUra was kindly provided by Robugen GmbH (Esslingen,West Germany). BVdUrd was synthesized according to Jones ef a/. (23).BVUra was prepared as previously described (24). 6-AThy was obtainedby alkaline cyclization of a-methylcyano-acetylurea according to theprocedure of Bergmann and Johnson (25). [mef/7y/-14C]Thy (60 mCi/mmol) and 5-fluoro-[6-14C]Ura (55 mCi/mmol) were purchased from

Amersham (Buckinghamshire, United Kingdom).In Vitro Pyrimidine Degradation. A 105,000 x g rat liver extract was

prepared according to the procedure of Shiotani and Weber (12); thisextract was dialyzed overnight against 0.25 M sucrose in 35 HIM potassium phosphate buffer (pH 7.4), 5 mw 2-mercaptoethanol, and 2.5 mw

MgCI2. Thy and FUra degradation was assayed by a radiochemicalmethod involving production of radioactive catabolites from [14C]Thy and[14C]FUra. The assay mixture contained 20 Â¿Â¿Mof radioactive Thy or

FUra, 250 IÂ¿MNADPH, 35 rriM potassium phosphate, and liver extract(0.5 mg protein/ml of reaction mixture). After incubation at 37Â°C, the

reaction was stopped by 30% perchloric acid; precipitated proteins wereeliminated by centrifugation. After neutralization, 10 /*! of the supernatantwere spotted onto Merck cellulose (for Thy) or silica gel (for FUra)-coated

plates. Ascending chromatography was carried out using as eluents amixture of fert-butyl alcohol:methylethylketone:water:ammonium hydroxide (40:30:30:10) (26) for cellulose plates or a mixture of chloro-

form:methanol:acetic acid (30:10:5) (27) for silica gel plates. The plateswere scanned with a thin layer radiochromatogram scanner to locateand integrate the radioactive products. Inhibition studies of pyrimidinedegradation by BVUra were performed under the same conditions butwith different concentrations of inhibitor as described in the legends tothe charts. From the crude liver extracts, H2Thy dehydrogenase waspartially purified by acid precipitation, ammonium sulfate fractionation,and DEAE-cellulose column chromatography (12); this last step enabled

us to obtain part of H2Thy dehydrogenase free from the two subsequentenzymes of the reductive catabolic pathway.

Pharmacokinetics in the Rat. Nucleosides and bases were administered i.p. to adult male Wistar rats weighing approximately 400-500 g.

The schedule or administration is described in the legends to the charts.The methods for plasma sample preparation and nucleoside and basequantitation by HPLC have been described previously (22). AUC (Â¿iMXh)for the plasma Thy and FUra concentrations were determined by thetrapezoidal rule from time 0 to the last data point by first order extrapolation to infinite time. The same procedure was followed for the urineThy and FUra concentrations.

In Vivo Inhibition of Pyrimidine Degradation by BVUra. BVUra (200Â¿imol/kg)was injected Â¡.p.in rats 1 h before they were sacrificed. Thelivers were excised and 105,000 x g supernatants were prepared anddialyzed as described above. H2Thy dehydrogenase activity was assayedin these supernatants. In vivo inhibition of H2Thy dehydrogenase byBVUra was followed by measuring plasma concentrations of Thy or FUrain rats pretreated with BVUra or BVdUrd.

Potentiation of FUra Effects in Leukemic Mice. P388 cells wereused for the in vivo studies on the antitumor activity of FUra. These cellswere kindly provided by Dr. A. A. Ovejera of the National Cancer Institute

Fig. 1. Structure of (E)-5-(2-bromovinyl)uracil.

and were maintained by weekly serial passage in DBA/2 mice. Anequivalent number of male and female mice were used in each set ofexperiments. DBA/2 mice were inoculated i.p. with 106 P388 cells on

day 0, and the compounds were administered i.p. at days 1, 3, 5, and 7after tumor cell inoculation. BVdUrd, FdUrd, and FUra were dissolved inEagle's minimum essential medium and injected in a volume of 200 or400 n\; BVUra was injected as a suspension in 400 /Â¿IEagle's minimum

essential medium. For potentiation assays, BVUra or BVdUrd were given60 or 90 min before FUra or FdUrd administration. The doses used inthese experiments were 2.5, 5, and 20 mg/kg for FUra, 10, 50, 200 mg/kg for FdUrd, and 200 ^mol/kg for BVUra and BVdUrd. For each individualmouse the day of death was recorded and from these data the meansurvival time was determined. Statistical analysis was based on Student's t test.

RESULTS

Pyrimidine Catabolism by Liver Extracts. The in vitro catab-olism of BVUra was compared with that of Thy and FUra. Forthese experiments, liver extracts were used, since the liverappears to be the major site for pyrimidine catabolism in vivo (7-9). When Thy was incubated with crude liver extract in thepresence of NADPH (under the conditions described in "Materialsand Methods"), it was rapidly transformed to 0-ureidoisobutyric

acid and 0-aminoisobutyric acid with an initial velocity of 35 nmol/

h/mg protein. No H2Thy was detected, which suggests that thereductive conversion of Thy to H2Thy is the limiting step in thecatabolism of Thy. When assayed under the same conditions,FUra was degraded twice as fast as Thy (70 nmol/h/mg protein),a-fluoro-ÃŸ-ureidopropionic acid and a-fluoro-/3-alanine were detected in the incubation medium while 5-fluorodihydrouracil was

not. When Thy or FUra was incubated with H2Thy dehydrogenase separated from dihydropyrimidine cyclohydrolase and N-carbamoyl-ÃŸ-alanine aminohydrolase by DEAE-cellulose chromatography, only the formation of H2Thy or 5-fluorodihydrouracil

could be demonstrated. In contrast with Thy or FUra, BVUrawas not degraded when incubated with either crude liver extractor partially purified H2Thy dehydrogenase.

In Vitro Inhibition of Pyrimidine Degradation by BVUra.BVUra not only was not a substrate of H2Thy dehydrogenasebut acted as an inhibitor of this enzyme. Indeed, when liverextract was preincubated at 37Â°Cwith BVUra, the degradation

of both Thy and FUra was significantly reduced; the rate ofdegradation of both Thy and FUra decreased as a function ofthe BVUra concentration in the incubation medium (Fig. 2). A50% inhibition of the degradation of 20 UM Thy or FUra wasobtained at a BVUra concentration of 2-3 /UM.An identical 50%inhibitory dose was obtained for BVUra if partially purified H2Thy-

dehydrogenase was used instead of crude liver extract.The inhibition of pyrimidine degradation was markedly en

hanced if BVUra was preincubated with the enzyme extract inthe presence of NADPH (Table 1A). Indeed, when BVUra wasadded at the same time as FUra to the incubation medium,inhibition of FUra degradation occurred only at 23% of thatobtained when BVUra had been preincubated with enzyme extract at 37Â°Cin presence of NADPH. When NADPH was omitted

from the preincubation medium, inhibition was only 21% of themaximal inhibition indicating that in the absence of NADPHBVUra did not bind to the enzyme. Moreover, the binding ofBVUra to the enzyme in the presence of NADPH was markedlyreduced when the preincubation was done at 4Â°C, since the


1095




BVUra (jjM)

Fig. 2. Inhibitory effects of BVUra on the degradation of Thy and FUra by liverextract. The 105,000 x g fraction of liver extract was preincubated for 10 min at37Â°Cwith BVUra at different concentrations and then assayed for Thy and FUradegradation as described in "Materials and Methods." The percentage of inhibition

was determined from the velocities of degradation obtained in the presence andabsence of BVUra.

was increased to 100 MM,it interfered with the binding of BVUrato the enzyme, since, under these conditions, the inhibition wasonly 7%. Thus, inhibition of H2Thy dehydrogenase occurred onlywhen BVUra was preincubated with the enzyme extract in thepresence of NADPH and was not reversed by extensive dialysis.These results are consistent with an irreversible inhibition ofH2Thy dehydrogenase by BVUra, whereby BVUra competes withFUra for a common binding site. Kinetics analyses are in progressto confirm this hypothesis. BVdUrd did not show any inhibitoryactivity under conditions where it could not be degraded toBVUra, i.e., by pyrimidine nucleoside phosphorylases that mayoccasionally be present in H2Thy dehydrogenase preparations.

In Vivo Clearance of Thy, FUra, and BVUra. When Thy orFUra were administered i.p. to rats at 200 ^mol/kg, Thy or FUraappeared in the plasma within a few minutes but were rapidlycleared from the bloodstream according to a first order process(Fig. 3) with a half-time of 22 and 14 min, respectively (Table 2).

Similarly, when the pyrimidine deoxynucleosides dThd or FdUrdwere administered Â¡.p.,they also appeared in the blood within afew minutes and were then rapidly eliminated from the circulation.

Table 1

Erteci of preincubation of the enzyme preparation with BVUra on the inhibition ofFUra degradation

In A, the enzyme extract was preincubated for 10 min at 37Â°Cwith or without

BVUra and/or NADPH. At the end of this period FUra (20 MM),and in some casesNADPH and BVUra, were added to the reaction mixture. The enzyme activity wasdetermined as previously described and the percentage of inhibition was determinedin comparison to the control sample. In B, the enzyme extract was preincubatedfor 10 min at 37Â°Cwith or without BVUra and/or NADPH; the mixture was then

dialyzed extensively against 35 mu phosphate buffer (pH 7.4) and used as enzymesource in the FUra degradation assay. Activity of the control sample was 47 nmol/h/mg protein.

Preincubation mixture

BVUra (4 MM) NADPH (250 MM) % Inhibition

17..e16a

7624"

Preincubation mixture

B BVUra (20 MM) NADPH (250 MM)

oo

86Ie

* NADPH added after preincubation." Activity of control sample, 73 nmol/h/mg protein.c BVUra added after preincubation." Preincubation at 4Â°Cinstead of 37Â°C.8 With FUra at 100 MMin the preincubation medium.

inhibition obtained with preincubation at 4Â°Cwas only 31% ofthat obtained with preincubation at 37Â°C.

These experiments indicated that BVUra might be irreversiblylinked to H2Thy-dehydrogenase. This hypothesis was verified by

dialyzing the enzyme extract after preincubation with 20 ^MBVUra with or without NADPH or FUra, and assaying the remaining activity with FUra as substrate (Table 1B). When theenzyme was preincubated at 37Â°Cwith BVUra in the absence

of NADPH and then extensively dialyzed, no inhibitory activitywas observed. If NADPH was present in the preincubationmedium before dialysis, the degradation of FUra was inhibitedby 86%. If the concentration of FUra in the preincubation mixture

0 2 i. 60 2 i. 6

Hours following administration of either Thy or FUra

Fig. 3. Effects of BVUra on the clearance of Thy and FUra from the plasma ofrats. Thy and FUra were administered i.p. at 200 Mmol/kg. Their plasma concentrations were evaluated at the indicated times after they had been administered alone(O) or 1 h after a previous adminstration of BVUra, 200 Mmol/kg (â€¢).Bars, one SD.The number of animals used are indicated in Table 2.

Table 2

Pharmacokinetics of Thy and FUra in the plasma of rats

Each compound was administered i.p. at 200 Mmol/kg according to the schedules described in Figs. 3 and 4. Mean Â±SD for f1/2and AUC were calculated fromthe individual values obtained for each rat. The increases in f1/2or AUC are indicatedin parentheses.

AdministrationThy

aloneBVUra +ThydThd

aloneBVUra + dThdBVdUrd + dThdNo.

ofrats6

34

53Plasma

Thyfi/2

(min)22Â±5(x

1)232 Â±47 (x10.5)14Â±3(X1)

206 Â±47 (x 14.7)199Â±23(x14.2)AUC

(MMxh)171

Â±26(x 1)1569 Â±558 (x9.2)50Â±15(x

1)1063 Â±321 (X21.3)

861Â±135(X17.2)Plasma

FUraFUra

aloneBVUra +FUraFdUrd

aloneBVUra + FdUrdBVdUrd + FdUrd7

43

33fi/z

(min)14Â±5(x

1)74 Â±12 (x5.3)21

Â±12(x 1)82 Â±8 (x 3.9)77 Â±7 (x 3.7)AUC

(MMxh)66Â±13(x

1)534 Â±92 (x8.1)44Â±10(x

1)532Â±83(x 12.1)480 Â±65 (x 10.9)


1096




The corresponding bases resulting from the phosphorolysis ofthe deoxynucleosides appeared in the plasma and were eliminated within 2-3 h (Fig. 4). Ura, 5-iodouracil, and 5-trifluoro-

methyluracil followed the same clearance pattern as Thy andFUra (data not shown).

On the contrary, after i.p. administration at 200 Mmol/kg,BVUra was eliminated from the plasma very slowly, so that itmaintained a concentration of 50-70 MMfor at least 6 h. BVUra

was then cleared from the plasma according to an apparent firstorder reaction with a mean half-life of about 8 h. Consequently,BVUra was still present in the plasma at a concentration of 5-

20 MM24 h after its administration. The mean AUC for BVUra inthe plasma was about 1000 MMXh. Following i.v. administrationat 200 Mmol/kg, BVUra reached a plasma concentration of 100MMat 20 min; the first order elimination gave a half-life of 7.5 h

and an AUC of 1175 MMXh. When BVdUrd was administered i.p.to rats it was rapidly transformed to BVUra upon the action ofpyrimidine nucleoside phosphorylases (22). Consequently,BVUra reached a plasma concentration of 70 MM 2-4 h afterBVdUrd administration and was then eliminated with a half-life

of 7.5 h; its mean AUC was 1075 MMXh. After i.v. administrationof BVdUrd, a maximal concentration of 60 MM BVUra wasreached within 2-4 h; the half-life of BVUra was about 7.1 h and

its plasma AUC was 782 MMXh.The peculiar behavior of BVUra most probably resulted from

its resistance to the action of H2Thy dehydrogenase. Eliminationof BVUra from the circulation by the kidney was low. The amountof BVUra eliminated in the urine during the first 48 h followingadministration of BVUra accounted for only 1.5-10% of the

amount of BVUra administered. The maximum renal excretion ofBVUra occurred between 4 and 24 h after its administration andreached peak urinary concentration of 700 MM.After 48 h, at atime when BVUra could not longer be detected in the plasma,low concentrations of BVUra were still present in the urine.

HPLC analyses demonstrated the presence of BVdUrd and(E)-5-(2-bromovinyl)uridine in the urine as identified by their re

tention time and by the action of pyrimidine nucleoside phosphorylases. Since these two products were not present in the plasmaat the time they were detected in the urine, it could be assumedthat they were formed by a pentosyl exchange reaction at thekidney level.

in Vivo Inhibition of Pyrimidine Degradation by BVUra. The

0 2 l> 0 2 I,

Hours following administration of cither dThdor FdUrd

Fig. 4. Effects of BVUraand BVdUrdon the plasmaclearanceof Thy and FUragenerated from the cleavage of their corresponding deoxynucleosides in rats.PlasmaThy and FUraconcentrations were determined at the indicatedtimes afteri.p. administrationof dThd or FdUrd at 200 >imol/kg,without (O)or with a previousadministration of BVUra, 200 Â¿imol/kg(â€¢)or BVdUrd (â€¢),3 h before the injectionof dThd or FdUrd.Bars, one SO.

inhibitory effect of BVUra on pyrimidine degradation in vivo wasfirst demonstrated by comparing the H2Thy dehydrogenase activity in the dialyzed 105,000 x g fractions of livers removedfrom rats pretreated with BVUra (200 Mmol/kg) to the livers fromcontrol rats. The degradation of FUra was inhibited by 97% inliver extracts from rats pretreated with BVUra as compared toliver extracts from untreated rats.

In vivo inhibition of the catabolism of Thy and FUra was furtherdemonstrated by monitoring the effect of BVUra on the plasmaconcentrations and pharmacokinetic parameters of Thy andFUra. When BVUra was administered at 200 Mmol/kg 1 h beforethe i.p. injection of Thy or FUra at 200 Mmol/kg, plasma concentrations, i* and AUC were increased (Fig. 3; Table 2). Similarly,plasma concentrations, tvâ€žand AUC of Thy and FUra, generatedby the phosphorolytic cleavage of dThd and FdUrd, were increased when these nucleosides were adminstered 3 h afterBVUra (Fig. 4; Table 2). When BVdUrd instead of BVUra wasadministered, the plasma concentrations, f,/,, and AUC of Thyand FUra, generated from dThd and FdUrd, respectively, againwere markedly increased (Fig. 4; Table 2). Other pyrimidinebases, which are substrates of H2Thy dehydrogenase such asUra, 5-iodouracil, and 5-trifluoromethyluracil also showed a

marked increase in plasma, f,/,, and AUC following administrationof BVUra or BVdUrd (data not presented).

Inhibition of pyrimidine degradation was noted at 8, 18, 24,and even 48 h after a single BVUra (200 Mmol/kg) administration,although at 48 h BVUra was undetectable in the plasma. A doseof BVUra, 25 Mmol/kg, was as efficient as a dose of 200 Mmol/kg, and BVUra, even at 3 Mmol/kg, reached 80% of its maximuminhibitory effect. At lower doses the inhibitory effect rapidlydeclined.

In Vivo Enhancement of the Antileukemic Effects of FUraby BVUra. Since BVUra protected FUra from degradation notonly in vitro but also in vivo, BVUra might be expected to enhancethe antitumor activity of FUra. This turned out to be the case asillustrated by the experiments shown in Fig. 5. When DBA/2mice were inoculated i.p. with 1 million P388 leukemia cells, theydied within 10 days after tumor cell inoculation. When FUra wasadministered at either 2.5 or 5 mg/kg at days 1,3,5, and 7 after

ra 15

'

o-1-

ii*l

â€¢;

r

25 5 20FUra Img/kg!

0 10 200FdUrd /mg/kg)

Fig. 5. Effect of a previous administrationof BVUraon the antileukemicactivityof FUraand FdUrdinmicebearingP388leukemiacells.DBA/2 micewere inoculatedi.p. with 1 million P388 leukemia cells at day 0. At days 1, 3, 5, and 7 after cellinoculation,the mice were treated i.p. with varyingdoses of FUraor FdUrd,withoutP) or with (H) administration of BVUra (200 ^mol/kg) 1 h before FUra or FdUrd.For each treatment group the mean day of death Â±SD (bars) is presented.Numbers in parentheses, number of mice in each group.


1097




tumor cell inoculation, it increased the survival of mice to 118and 128%, respectively, as compared to the control group (Table3). However, when BVUra (200 /Â¿mol/kg)was administered 1 hbefore each FUra injection, the survival was further increased to150 and 176%, respectively. With these FUra doses, survivalsof BVUra-treated mice were significantly increased in comparisonto those of mice receiving only FUra (Table 4). At 20 mg/kg, FUraalone increased the mean survival time of leukemic mice to158%, and if this dose of FUra was preceded by BVUra at adose of 200 ^mol/kg, the life span decreased to that of thecontrol mice. When compared to that of mice receiving FUra at20 mg/kg alone, the survival of BVUra-pretreated mice receiving

FUra at 20 mg/kg was considerably decreased (Table 4). Clearly,this treatment regimen was toxic for the mice, as particularlydemonstrated by a marked loss of weight. BVUra administered

Table 3

Effects of FUra, FdUrd, and inhibitors ofpyrimidine degradation on the survival ofmice bearing P388 leukemic cells

DBA/2 mice inoculated i.p. at day 0 with 1 million P388 cells received at days1,3.5, and 7 an i.p. administration of the compounds listed below. Control micereceived only saline. Survival times are presented as the mean Â±SD and inpercentage as compared to the control group. The number of animals per group isindicated in parentheses.

CompoundNoneFUra

(2.5 mg/kg)FUra (5 mg/kg)FUra (20mg/kg)FdUrd

(10 mg/kg)FdUrd (50 mg/kg)FdUrd (200mg/kg)BVUra

(200 >imol/kg)BVdUrd (200 /Â¿mol/kg)DUra (200 /^mol/kg)DUra (50 ^mol/kg)EtUra (200 ^mol/kg)6-AThy (70 /imol/kg)Survival

(days)9.7

Â±0.8(48)1

1.4 0.4 (6)12.4 1.3(24)15.31.5(24)10.4

1.0(12)11.7 1.3(12)15.9 Â±1.4(12)9.2

Â±0.9 (6)10.2 Â±0.8 (6)12.5 Â±2.9 (6)13.1 Â±0.8(6)9.8 Â±0.8 (6)9.4 Â±0.6 (6)%

ofcontrol100118a

128a158a107"

121a164Â°94A1056

129a135"1016

97o"

Significantly different (P < 0.001) from control.6 Not significantly different (P > 0.05) from control.

Table 4

Influence of inhibitors of pyrimidine degradation on the effects of FUra and FdUrdon the survival of leukemic mice

DBA/2 mice bearing P388 leukemic cells (see Table 3) received at days 1, 3,5,and 7 an i.p. injection of FUra or FdUrd (at the indicated doses), 1 h after the i.p.administration of a H2Thy dehydrogenase inhibitor. Control mice received FUra orFdUrd only. Results are expressed as the percentage of survival compared to thecorresponding control group.

Inhibitors of pyrimidine degradation

Oimol/kg)None

BVUra (200)BVdUrd (200)DUra (200)DUra (50)EtUra (200)6-AThy(70)None

BVUra (200)BVdUrd (200)2.5100

150.2a151.1a10100

148a165.5aFUra

(mg/kg)5100

137.4a141.1a86"

141.1a98.8"

106.2"FdUrd

(mg/kg)50100

79.3a20100

60.5a60.5a37.6a

65.4a200100

54a48.3a

alone at 200 Mffiol/kg had neither antitumor nor apparent toxiceffects (Table 3).

FdUrd at 10 mg/kg had a very slight antileukemic effect whichwas considerably increased (mean survival time, 159%) whenFdUrd administration was preceded by BVUra; FdUrd alone at200 mg/kg was well tolerated and increased the mean survivaltime of mice to 164% in comparison to the control group butbecame toxic (as demonstrated by a marked loss of weight) ifBVUra had been injected previously; in this case the survivaltime was lower than for the control group (Fig. 5). The survivaltime of mice pretreated with BVUra (as compared to that of micereceiving FdUrd only) was considerably increased at low dosesof FdUrd while it was significantly decreased at high doses ofFdUrd (Table 4).

Almost identical effects were obtained if BVdUrd (200 u.mo\lkg) instead of BVUra was injected 90 min before the administration of FUra or FdUrd (Fig. 6; Table 4). The time interval of 90min was chosen, since after 90 min BVdUrd was almost completely cleared from the circulation and replaced by BVUra.

The enhancing effect of BVUra and BVdUrd on the antitumoractivity and toxicity of FUra in DBA/2 mice could be attributedto an inhibition of the degradation of FUra and hence an increasein the therapeutically active concentrations of FUra. Indeed, theplasma concentrations of FUra in mice, as in rats, were considerably increased if the mice had received BVUra or BVdUrdbefore FUra (Table 5).

In Vivo Effects of Some Other Inhibitors. We have alsoexamined the effects of in vivo pyrimidine degradation of someother inhibitors of H2Thy-dehydrogenase, i.e., DUra (16,18,19),

â€¢o 15

10

C-

1Â¿M

IS

10

0-0 25 B 20 O 10 50 200

fÃ¼rilmg/kgl FdUrd Img/kg)

Fig. 6. Effect of a previous adminstration of BVdUrd on the antileukemic activityof FUra or FdUrd in mice bearing P388 leukemia cells. The experimental conditionsare identical to those explained in the legend to Fig. 5, except that BVdUrd (200Aimol/kg) instead of BVUra was administered 90 min before FUra or FdUrd.

Tables

Influence of BVUra and BVdUrd on plasma concentrations of FUra in mice

Plasma concentrations of FUra were determined 20 min after i.p. administrationof different doses of FUra to DBA/2 mice. BVUra or BVdUrd was administered i.p.at 200 nmd/kg 1 h before FUra.

* Significantly different (P < 0.001) from control.6 Not significantly different (P > 0.05) from control.

FUra(mg/kg)5

1020

50Plasma

concentrations of FUra (>IM)upon injectionofFUra

alone1(13.4)a

22.5 (59)4BVdUrd

+FUra21

38.579

308BVUra

+FUra26(61.5)

54159(189)273

Numbers in parentheses, rat plasma values.


1098




EtUra (14), and 6-AThy (28). When injected i.p. in rats at 200/jmol/kg, DUra increased by 15- and 23-fold, respectively, theplasma half-life and AUC of FUra, while EtUra and 6-AThy onlyslightly increased the half-life and AUC of FUra. DUra, EtUra, and6-AThy were also investigated for their enhancing effects on theantileukemic activity of FUra in mice (Table 4). Only DUra at 50Â¿Ãinol/kgcaused a marked increase in the survival time of leu-

kemic mice over that achieved by FUra alone. Unlike BVUra,DUra had itself some antileukemic activity (Table 3), and whenused at 200 ^mol/kg in conjunction with FUra (20 mg/kg) iteffected a dramatic decrease in the mean life span of the mice(Table 4).

DISCUSSION

The present results demonstrate that BVUra is more slowlycleared from the bloodstream than the other pyrimidine basesprobably because it is not a substrate for the first enzyme of thereductive pathway for pyrimidine degradation, i.e., H2Thy dehydrogenase. Indeed, BVUra is not degraded by liver extractswhich are rich in H2Thy dehydrogenase (7-9) or by partially

purified enzyme. In vivo, it takes 48 h before BVUra is entirelycleared from the bloodstream after it has been administered torats i.p. at 200 /Â¿mol/kg.The plasma AUC values for BVUraobtained after i.p. or i.v. adminstration of BVUra (1000 and 1175MMXh, respectively) are clearly higher than those obtained forThy (171 and 222 /Â¿wxh,respectively) or FUra (66 and 144MMXh, respectively), indicating that BVUra is more slowly clearedfrom the bloodstream than Thy and FUra.

BVUra is only partially eliminated by renal clearance. Althoughhigh concentrations (up to 700 Â¿JM)of BVUra were found in theurine within the first 24 h following administration of BVUra,urinary BVUra did not represent more than 10% of the administered BVUra. Possibly, BVUra could be transformed in vivo toBVdUrd by pyrimidine nucleoside phosphorylases (22) then phos-

phorylated by cellular kinases and incorporated into nucleic acids.However, BVdUrd is not a substrate for cellular cytosol dThdkinase; it only is a good substrate both for the mitochondrialdThd kinase and herpes simplex and varicella-zoster virus-encoded enzymes (29).5 Thus, in uninfected cells or tissues BVdUrd

can be phosphorylated only by mitochondrial dThd kinase. Towhat extent if any this phosphorylation contributes to the clearance of BVUra from the circulation is not clear, however. Thereare various other metabolic pathways or other elimination routes(e.g., biliary, fecal) by which BVUra may be eliminated, and inaddition, BVUra may be bound to and retained by plasma ortissue proteins.

The present results also demonstrate that BVUra acts as apowerful inhibitor of H2Thy dehydrogenase and thereby inhibitsthe degradation of other pyrimidines that are substrates for thisenzyme both in vitro and in vivo. In vitro, BVUra reduces thedegradation of Thy and FUra (Fig. 2). The findings that (a)preincubation with liver extract is required for the inhibitory effectof BVUra on H2Thy dehydrogenase activity, (b) this inhibitoryeffect is not reversed by extensive dialysis, and (c) in vivoinhibition of pyrimidine degradation is maintained after BVUrahas been completely cleared from the circulation, suggest that,like DUra (16), BVUra may be an irreversible inhibitor of H2Thy

5Unpublished observations.

dehydrogenase. This enzyme is a flavoprotein (12). It has beendemonstrated (see Ref. 30 for review) that several flavin-depend

ent enzymes can be inhibited irreversibly by substrate analogueswhich contain a function which is converted to a highly reactivegroup within the active site. This highly reactive group may theninteract with an essential protein residue or prosthetic groupbefore dissociation occurs, thereby causing irreversible inhibition.Although the bromovinyl is per se less reactive than the diazogroup (31, 32), its reactivity may be enhanced considerably inthe presence of NADPH, since this cofactor is necessary duringthe preincubation period to ensure irreversible binding of BVUrato the enzyme.

BVUra inhibits the degradation of Thy and FUra in vivo. Whenrats are pretreated with BVUra before the administration of Thyor FUra, plasma AUC of Thy and FUra (Table 2) is considerablyincreased (9.2- and 8.1-fold, respectively). Under these condi

tions, BVUra probably acts by blocking the H2Thy dehydrogenase since the activity of this enzyme is markedly suppressed inliver extracts from rats pretreated with BVUra. Based on itsinhibitory effect on pyrimidine degradation BVUra may be expected to increase the in vivo therapeutic activity of 5-substituted

Ura analogues such as FUra. In the experimental model used inthis study, BVUra enhanced the antitumor activity of FUra, sothat FUra, at 5 mg/kg, in combination with BVUra was moreeffective than FUra alone at 20 mg/kg (Fig. 5). The increasedtherapeutic activity of FUra in leukemic mice with BVUra isprobably due to an inhibition of FUra degradation, since in mice,like in rats, FUra plasma concentrations are considerably increased following BVUra pretreatment; yet, an exact correlationbetween plasma FUra concentration and increase in survival timecould not be established (Table 5; Figs. 5 and 6). Further studies,i.e., determination of peritoneal fluid concentrations of FUra maybe necessary to evaluate such a correlation.

FdUrd at 10 mg/kg, in the presence of BVUra, has approximately the same antitumor activity as FdUrd alone at 100 mg/kg. It has been previously demonstrated that BVUra does notinfluence the cleavage of pyrimidine deoxynucleosides to thefree pyrimidines (22); thus, the enhancing effect of BVUra on theantitumor activity of FdUrd is probably due to an inhibition ofFUra degradation following the cleavage of FdUrd to FUra. In therat, FdUrd is rapidly transformed to FUra, which attains lowerplasma concentrations than those achieved by the administrationof FUra itself (Table 2). However, following a previous BVUraadministration, the plasma FUra concentration achieved uponequimolar administration of FUra and FdUrd are almost identical.This may explain why FUra at 5 mg/kg and FdUrd at 10 mg/kghave approximately the same antileukemic activity in the presence of BVUra, whereas in the absence of BVUra, FdUrd dosesof 100 mg/kg are needed to achieve the same antitumor effectas FUra at 20 mg/kg. Plasma AUC values estimated for FUrafrom the data of Table 2 (51, 103, 89, and 108 jiMXh uponadministration of FUra, 20 mg/kg; FUra, 5 mg/kg plus BVUra;FdUrd, 100 mg/kg; and FdUrd, 10 mg/kg plus BVUra, respectively) correspond to a maximal increase in survival, while AUCvalues obtained with FUra, 20 mg/kg plus BVUra and FdUrd,100 mg/kg plus BVUra (410 and 1081 Â¿tMXh,respectively) correspond to a toxic effect. Thus, antitumor activity as well astoxicity of FdUrd and FUra seem to be determined by the AUCof FUra.

The relatively weak activity of FdUrd in the P388 tumor model


1099




may Â¡npart be attributed to its own metabolism by the P388cells, which are, in contrast to other tumor cells, rather poor inthymidine kinase activity (33). This would limit the phosphoryla-tion of FdUrd to 5-fluoro-2'-deoxyuridine-5'-monophosphate in

these cells and consequently the inhibitory action of FdUrd onthymidylate synthetase (34) and its incorporation into DNA (35).P388 cells also have little pyrimidine nucleoside phosphorylaseactivity (33, 35) which means that they are virtually unable toconvert FUra to FdUrd or 5-fluorouridine. However, FUMP may

be formed directly from FUra upon the action of pyrimidinephosphoribosyl transferase which appears to be quite active inP388 cells (33, 36). Moroever, Mulkins and Heidelberger (33)have demonstrated that P388 cells resistant to FUra have areduced pyrimidine phosphoribosyl transferase activity in comparison to the wild-type cells. Thus, the inhibitory effects of FUra

on P388 cells may be related to the conversion of FUra to FUMPand the eventual incorporation of FUMP into RNA (37). Accordingto our observations BVUra could act by blocking the degradationof FUra, thereby making available greater amounts of FUra to beprocessed via pyrimidine phosphoribosyl transferase to FUMP,which may then give rise to an increased incorporation of FUrainto RNA of P388 cells.

The inhibition of Thy and FUra degradation achieved in vivowhen Thy and FUra are administered 3 h after the injection ofBVdUrd, i.e., a time when BVdUrd has been completely converted to BVUra in the plasma (22), is apparently mediated byBVUra, although theoretically, it could also be attributed toBVdUrd itself. In vitro, BVdUrd partially inhibits the degradationof Thy and FUra by crude liver extracts. As demonstrated byHPLC analysis, however, BVdUrd, which is an excellent substrate for pyrimidine nucleoside phosphorylases (38, 39), is almost entirely transformed to BVUra by liver extracts. Liver isindeed a rich source of these enzymes (40). When the processof phosphorolysis is eliminated by suppression of phosphate inthe incubation medium or by the use of a partially purified enzymedevoid of pyrimidine nucleoside phosphorylase activity, inhibitionof pyrimidine degradation does not occur. These observationsconfirm previous findings that pyrimidine deoxynucleosides ofwhich the corresponding base may be either substrate or inhibitorof H2Thy dehydrogenase do not exert any effect on the enzymeactivity (14).

In vitro, DUra is a better inhibitor of FUra and Thy degradationthan is BVUra (inhibitory dose for DUra, 46-60 nw, as comparedto 2-3 fiM for BVUra). Among the inhibitors of pyrimidine degra

dation examined in this study (Table 4), only DUra potentiatedthe antineoplastic activity of FUra in vivo to a similar degree asBVUra. However, DUra, at 200 /tmol/kg was clearly toxic for themice during the period it was administered, although this toxicitydid not appear to affect the mean survival time. At 200 /Â¿mol/kg,DUra also increased the toxicity of FUra. At 50 Â¿imol/kg(approximately, 6.9 mg/kg), a concentration close to the 50% lethal dose(10 mg/kg/day for 4 days) for Fischer rats (18), DUra was slightlybetter tolerated but still showed some signs of toxicity at thebeginning of the treatment period. As previously described (41),DUra has by itself some antileukemic activity (increase of meansurvival time to 135%), which makes it more difficult to correctlyinterpret its potentiating effect on the antitumor activity of FUra.In contrast with DUra, BVUra has no antileukemic activity of itsown (Table 3). No toxicity studies have been conducted withBVUra. However, its predecessor, BVdUrd has been the subject

of toxicity studies, and these have shown that BVdUrd does notlead to untoward effects when administered i.p. to mice at 750-1500 /imol/kg daily for 4 consecutive wk (42). Since BVdUrd israpidly transformed to BVUra in vivo, it can be surmised thatBVUra has no marked toxicity at doses up to 1500 /imol/kg/day.

In conclusion, in addition to its ability to serve as a precursorfor the generation of the potent and selective antiviral drugBVdUrd in vivo (22), BVUra also appears to be a powerful inhibitorof the degradation of pyrimidines (such as FUra) via the H2Thydehydrogenase pathway, allowing the use of 4- to 8-fold lower

doses of FUra to achieve an equivalent therapeutic effect in theP388 leukemia mouse model.

ACKNOWLEDGMENTS

The authors thank I. Belloc, F. Dupuch, M. Verstreken for their expert technicalassistance and C. Callebaut, N. Grillon and L. Palmaerts for their valuable help inthe preparation of the manuscript.

REFERENCES

1. Wastemack, C. Degradation of pyrimidines and pyrimidine analogsâ€”pathwaysand mutual influences. Pharmacol. Ther., 8: 629-651, 1980.

2. Fink, K., Henderson, R. B., and Fink, R. M. /i-Aminoisobutyric acid in rat urinefollowing administration of pyrimidines. J. Biol. Chem., 797: 441-452,1952.

3. Fink, R. M., Fink, K., and Henderson, R. B. (j-Amino acid formation by tissueslices incubated with pyrimidines. J. Biol. Chem., 201: 349-355,1953.

4. Fink, R. M., McGaughey, C., Cline, R. E., and Fink, K. Metabolism of intermediate pyrimidine reduction products in vitro. J. Biol. Chem., 278:1-8,1956.

5. Canellakis, E. S. Pyrimidine metabolism. I. Enzymatic pathways of uracil andthymine degradation. J. Biol. Chem., 227: 315-322,1956.

6. Fritzson, P. Properties and assay of dihydrouracil dehydrogenase of rat liver.J. Biol. Chem., 235: 719-725, 1960.

7. Fritzson, P. The relation between uracil-catabolizing enzymes and rate of ratliver regeneration. J. Biol. Chem., 237: 150-156, 1962.

8. Queener, S. F., Morris, H. P., and Weber, G. Dihydrouracil dehydrogenaseactivity in normal, differentiating and regenerating liver and in hepatomas.Cancer Res., 37: 1004-1009.1971.

9. Barrett, H. W., Munavalli, S. N., and Newmark, P. Synthetic pyrimidines asinhibitors of uracil and thymine degradation by rat-liver supernatant. Biochim.Biophys. Acta, 97: 199-204, 1964.

10. Ho, D. H. W., Haebenen, R., Benjamin, R. S., and Bodey. G. P. Distribution ofdihydrouracil dehydrogenase in human tissues and its inhibition by diazouracil.Proc. Am. Assoc. Cancer Res.. 22: 25, 1981.

11. Dorsett, M. T., Morse, P. A., Jr., and Gentry, G. A. Inhibition of rat dihydropy-rimidine dehydrogenase by 5-cyanouracil in vitro. Cancer Res., 29: 79-82,1969.

12. Shiotani, T., and Weber, G. Purification and properties of dihydrothyminedehydrogenase from rat liver. J. Biol. Chem., 256: 219-224.1981.

13. Sebesta, K., Bauerova, J.. and Sormova. Z. Inhibition of uracil and thyminedegradation by some 5-substituted uracil analogues. Biochim. Biophys. Ada,50:393-394,1961.

14. Newmark, P., Stephens. J. D., and Barrett, H. W. Substrate specificity of thedihydro-uracil dehydrogenase and uridine phosphorylase of rat liver. Biochim.Biophys. Acta, 62: 414-416,1962.

15. Goedde, H. W.. Agarwal, D. P., and Eickhoff, K. Purification and properties ofdihydrouracil dehydrogenase from pig liver. Hoppe-Seyler's Z. Physiol. Chem.,

357.945-951,1970.16. Cooper, G. M., and Greer, S. Irreversible inhibition of dehatogenation of 5-

iodouracil by 5-diazouracil and reversible inhibition by 5-cyanouracil. CancerRes., 30: 2937-2941,1970.

17. Gentry, G. A., Morse, P. A., Jr., and Dorsett, M. T. In vivo inhibition of pyrimidinecatabolism by 5-cyanouracil. Cancer Res.. 37: 909-912,1971.

18. Cooper, G. M., Dunning, W. F., and Greer, S. Role of catabolism in pyrimidineutilization for nucleic acid synthesis in vivo. Cancer Res., 32: 390-397,1972.

19. Ferdinandus, J. A., and Weber, G. Effect of 5-diazouracil on the synthetic andcatabolic utilization of thymine in rat liver. Proc. See. Exp. Biol. Med., 739:592-596,1972.

20. Ensminger, W.. and Rosowsky, A. Prevention of methotrexate toxicity withthymidine esters or thymidine/thymine plus diazouracil. Proc. Am. Assoc.Cancer Res., 78:109,1977.

21. De Ctercq, E., Descamps, J., De Somer, P., Barr, P. J., Jones, A. S., andWalker, R. T. (E)-5-(2-Bromovinyl)-2'-deoxyuridine: a potent and selective anti-

herpes agent. Proc. Nati. Acad. Sci. USA, 76: 2947-2951,1979.22. Desgranges, C., Razaka, G., Drouiltet, F., Bricaud, H., Herdewijn, P., and De

Clercq, E. Regeneration of the antiviral drug (E)-5-(2-bromovinyl)-2'-deoxyuri-


1100




dine in vivo. Nucleic Acids Res., 12: 2081-2090,1984.23. Jones, A. S., Verfielst, G., and Walker, R. T. The synthesis of the potent anti-

herpes agent, E-5(2-bromovinyl)-2'-deoxyuridine and related compounds. Tet

rahedron Lett., 4415-4418, 1979.24. Barr, P. J., Jones, A. S.. Verfielst, G., and Walker, R. T. Synthesis of some 5-

halogenovinyl derivatives of uracil and their conversion into 2'-deoxyribonucle-

osides. J. Chem. Soc. Perkin Trans. 1,1665-1670,1981.25. Bergmann, W., and Johnson, T. B. Researches on pyrimidines. CXXXII. A new

synthesis of thymine. J. Am. Chem. Soc., 55: 1733-1735,1933.

26. Fink, K., Cline, R. E., Henderson, R. B., and Fink, R. M. Metabolism of thymine(methyl-C14 or -2-C14) by rat liver in vitro. J. Biol. Chem., 227: 425-433,1956.

27. ligo, M., Kuretani, K., and Hoshi, A. Relationship between antitumor effect andmetabolites of 5-fluorouracil in combination treatment with 5-fluorouracil andguanosine in ascites Sarcoma 180 tumor system. Cancer Res., 43: 5687-

5694,1983.28. Matthes, E., Barwolff, D., and Langen, P. Inhibition by 6-aminothymine of the

degradation of nucteosioes (5-iododeoxyuridine, thymidine) and pyrimidinebases (5-iodouracil, uracil and 5-fluorouracil) in vivo. Acta Biol. Med. Ger., 32:483-502,1974.

29. Cheng, Y-C., Dutschman, G., De Clercq, E., Jones, A. S., Rahim, S. G.,Verfielst, G., and Walker, R. T. Differential affinities of 5-(2-halogenovinyl)-2'-

deoxyuridines for deoxythymidine kinases of various origins. Mol. Pharmacol.,20:230-233,1981.

30. Ghisla, S., Wenz, A., and Thorpe, C. Suicide substrates as irreversible inhibitorsof flavoenzymes. In: U. Brokbeck (ed.), Enzyme Inhibitors, pp. 43-60. Basel:

Verlag Chemie, 1980.31. Previe, E., and Fister, G. Incorporation of 5-diazouracil-[2-'''C] into ribonucleic

acid of Escherichia coli during division inhibition. J. Bacteriol., 707: 188-195,

1970.32. Rando, R. R. On the mechanism of action of antibiotics which act as irreversible

enzyme inhibitors. Biochem. Pharmacol., 24: 1153-1160, 1975.33. Mulkins, M. A., and Heidelberger, C. Biochemical characterization of fluoro-

pyrimidine-resistant murine leukemic cell lines. Cancer Res., 42: 965-973,1982.

34. Ardalan, B., Cooney, D. A., Jayaram, H. N., Carneo, C. K., Glazer, R. l.,Macdonald, J., and Schein, P. S. Mechanisms of sensitivity and resistance ofmurine tumors to 5-fluorouracil. Cancer Res., 40:1431-1437,1980.

35. Schuetz, J. D., Wallace, H. J., and Diasio, R. B. 5-Fluorouracil incorporationinto DMA of CF-1 mouse bone marrow cells as a possible mechanism oftoxicity. Cancer Res., 44:1358-1363,1984.

36. Kessel, D., Hall, T. C., and Reyes, P. Metabolism of uracil and 5-fluorouracil inP388 murine leukemia cells. Mol. Pharmacol., 5: 481-486,1969.

37. Wilkinson, D. S., and Pilot, H. C. Inhibition of ribosomal ribonucleic acidmaturation in Novikoff hepatoma cells by 5-fluorouracil and 5-fluorouridine. J.Biol. Chem., 248: 63-68,1973.

38. Desgranges, C., Razaka, G., Rabaud, M., Bricaud, H., Balzarini, J. and DeClercq, E. Phosphorolysis of (Â£)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) andother 5-substituted 2'-deoxyuridines by purified human thymidine phosphoryl-

ase and intact blood platelets. Biochem. Pharmacol., 32: 3583-3590,1983.39. Liermann, B., and Herrmann, G. (E)-5-(2-Bromovinyl)-2'-deoxyuridine. A good

substrate for mammalian pyrimidine nucleoside phosphorylases. Biomed.Biochim. Acta, 42: K35-K38,1983.

40. Yamada, E. W. Pyrimidine nucleoside phosphorylases of rat liver. Separationby ion exchange chromatography and studies of the effect of cytidine or uridineadministration. J. Biol. Chem., 243: 1649-1655, 1968.

41. Sassenrath, E. N., Keils, A. M., and Greenberg, D. M. Characterization studieson the carcinostatic activity of 5-diazouracil. Cancer Res., 79: 259-267,1959.

42. Walker, R. T., Jones, A. S., De Clercq, E., Deseamos, J., Allaudeen, H. S.,and Kozarich, J. W. The synthesis and properties of some 5-substituted uracilderivatives. Nucleic Acids Res., Symposium Series No. 8, s95-s102,1980.

CANCER RESEARCH VOL.46 MARCH 1986

1101



1986;46:1094-1101. Cancer Res Claude Desgranges, Gabriel Razaka, Erik De Clercq, et al. Antitumor Activity of 5-Fluorouracil in Rats and Leukemic Mice

)-5-(2-Bromovinyl)uracil on the Catabolism andEEffect of (

Updated version

http://cancerres.aacrjournals.org/content/46/3/1094

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

[email protected] 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://cancerres.aacrjournals.org/content/46/3/1094To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



effect of (e)-5-(2-bromovinyl)uracil on the …...effect of (e)-5-(2-bromovinyl)uracil on the...

Documents