2-Bromovinyl)Uracil on the Catabolism and Antitumor Activity of 5-Fluorouracil in Rats and Leukemic Mice1

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

Effect of (E)-5-(2-Bromovinyl)uracil on the Catabolism and Antitumor Activity of 5-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©8de 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 INTRODUCTION In contrast to thymine and 5-fluorouracil (FUra) which were Thy4 and Ura are both subject to the same catabolic pathway cleared from the bloodstream within 2-4 h after their i.p. admin which is, in contrast to that of the purine bases, a reductive istration (200 Â¿imol/kg)to rats, (Â£)-5-(2-bromovinyl)uracil (BVUra) pathway (see Ref. 1 for review). The first step of the catabolism maintained a concentration of 50-70 U.Mfor at least 6 h and was of Thy and Ura is the reduction of the 5:6 double bond of the still present in the plasma 24 h after its administration. In vitro pyrimidine ring, followed by the cleavage of the 3:4 bond to give experiments with rat liver extracts indicated that BVUra was not 0-ureidoisobutyric acid and /3-ureidopropionic acid from which ÃŸ- a substrate but an inhibitor for the reductive step in pyrimidine amino acids are formed with the release of ammonia and carbon degradation catalyzed by dihydrothymine dehydrogenase. Ki dioxide (2-5). The first enzyme of this catabolic pathway, H2Thy netic and dialysis experiments suggested that BVUra was an dehydrogenase (EC 1.3.1.2), was identified as the rate-limiting irreversible inhibitor of this enzyme. The binding of BVUra to the enzyme (5-8). The liver appears to be the major site of pyrimidine enzyme depended on the presence of reduced nicotinamide catabolism (7-9); the activity of H2Thy dehydrogenase in the liver adenine dinucleotide phosphate in the reaction mixture. Dihydro is 20-fold higher than in lung, bone marrow, and colon (10). thymine dehydrogenase activity was also inhibited in the dialysed 5-Substituted Ura analogues and in particular 5-halogenated 105,000 x g supernatant fraction of livers from rats that had Ura are also substrates for this enzyme (11,12), and it is obvious previously been treated with BVUra. Such inhibitory effects also that the degradation of FUra via H2Thy dehydrogenase may occurred in vivo; previous adminstration of BVUra increased the affect the therapeutic efficacy of FUra in the treatment of cancer. plasma half-lives of thymine and FUra by 10- and 5-fold and their The use of H2Thy dehydrogenase inhibitors may be helpful not area under the curve by 9- and 8-fold, respectively. only for the elucidation of the mechanism of action of FUra and The effect of BVUra on the antitumor activity of FUra was its analogues but also with respect to an enhancement of their evaluated in DBA/2 mice inoculated with 106 P388 leukemia therapeutic activity. Some inhibitors have been described previ cells. The mean survival times for the control and FUra-treated ously; among the Ura analogues (9, 13-15), 5-cyanouracil (11, mice (5 mg/kg at 1, 3, 5, and 7 days after tumor cell inoculation) 16, 17) and DUra (10, 15, 16, 18-20) are the most potent were 9.7 and 12.4 days, respectively. When BVUra (200 /Â¿mol/ inhibitors of pyrimidine degradation in vitro or in vivo. Most of kg) was administered 1 h before each injection of FUra, the mean these compounds act in a competitive manner, since they are survival time was extended to 17.1 days. BVUra alone did not also substrates of H2Thy dehydrogenase (11-14). Some of them affect the mean survival time. When the dose of FUra was act in vivo, both by decreasing the degradation of Thy and FUra increased to 20 mg/kg, the mean survival time was 15.3 days; and by increasing their incorporation into DNA and RNA (18,19). upon a preceding injection of BVUra the mean survival time As a consequence, they not only enhance the antitumoral activity decreased to 9.2 days. The latter effect probably resulted from of FUra but also its toxicity (17,18). While we were studying the an increased toxicity of FUra. Similar results were obtained if catabolism of the potent and selective antiherpes agent, BVdUrd FUra was replaced by 5-fluoro-2'-deoxyuridine and BVUra by (21) in the rat, we found that its degradation product BVUra (Fig. (Â£)-5-(2-bromovinyl)-2'-deoxyuridine. The enhancement of both 1), in contrast to other pyrimidine bases, had a relatively long the antitumor and toxic effects of FUra by BVUra were most half-life in plasma (22). We have now pursued these observa probably due to an inhibition of FUra degradation, since, like in tions, and here we demonstrate that BVUra, being itself not rats, BVUra increased the plasma half-life of FUra in DBA/2 mice. active as a substrate, reduces the degradation of other pyrimi Hence BVUra appears to be an interesting compound, increasing dine bases, such as FUra, by inhibition of the H2Thy dehydro the potency of FUra by decreasing its degradation. genase activity in vivo and in vitro. Moreover, BVUra enhances the antitumor activity of FUra in the P388 leukemia model in Received 7/20/84; revised 10/28/85; accepted 11/22/85. ' This investigation was supported by grants from the Institut National de la DBA/2 mice. SantÃ©et de la Recherche MÃ©dicale,the University of Bordeaux II. the Belgian Fonds voor Geneeskundig Wetenschappelijk Onderzoek, and the Belgian Gecon- 4The abbreviations used are: Thy, thymine; Ura, uracil; H2Thy, dihydrothymine; certeerde Onderzoeksacties. FUra, 5-fluorouracil; DUra, 5-diazouracil; BVdUrd, (Â£)-5-(2-bromovinyl)-2'-deoxyu- 2 Recipient of a fellowship award from North Atlantic Treaty Organization. To ridine; BVUra, (E)-5-(2-bromovinyl)uracil; EtUra, 5-ethyluracil; 6-AThy, 6-aminothy- whom requests for reprints should be addressed. mine; AUC, area under the curve; FdUrd, 5-fluoro-2'-deoxyuridine; dThd, thymidine; 3 Senior Research Assistant of the Belgian National Fonds voor Wetenschap FUMP, 5-fluorouridine-5'-monophosphate; HPLC, high-performance liquid chro- pelijk Onderzoek. matography.

CANCER RESEARCH VOL. 46 MARCH 1986 1094

MATERIALS AND METHODS and were maintained by weekly serial passage in DBA/2 mice. An equivalent number of male and female mice were used in each set of experiments. DBA/2 mice were inoculated i.p. with 106 P388 cells on Chemicals. Thy, FUra, DUra, other Ura analogues, their corresponding deoxynucleosides and NADPH were obtained from Sigma Chemical Co., day 0, and the compounds were administered i.p. at days 1, 3, 5, and 7 St. Louis, MO. EtUra was kindly provided by Robugen GmbH (Esslingen, after tumor cell inoculation. BVdUrd, FdUrd, and FUra were dissolved in Eagle's minimum essential medium and injected in a volume of 200 or West Germany). BVdUrd was synthesized according to Jones ef a/. (23). BVUra was prepared as previously described (24). 6-AThy was obtained 400 n\; BVUra was injected as a suspension in 400 /Â¿IEagle's minimum by alkaline cyclization of a-methylcyano-acetylurea according to the essential medium. For potentiation assays, BVUra or BVdUrd were given procedure of Bergmann and Johnson (25). [mef/7y/-14C]Thy (60 mCi/ 60 or 90 min before FUra or FdUrd administration. The doses used in mmol) and 5-fluoro-[6-14C]Ura (55 mCi/mmol) were purchased from these experiments were 2.5, 5, and 20 mg/kg for FUra, 10, 50, 200 mg/ Amersham (Buckinghamshire, United Kingdom). kg for FdUrd, and 200 ^mol/kg for BVUra and BVdUrd. For each individual In Vitro Pyrimidine Degradation. A 105,000 x g rat liver extract was mouse the day of death was recorded and from these data the mean prepared according to the procedure of Shiotani and Weber (12); this survival time was determined. Statistical analysis was based on Stu dent's t test. extract was dialyzed overnight against 0.25 M sucrose in 35 HIM potas sium phosphate buffer (pH 7.4), 5 mw 2-mercaptoethanol, and 2.5 mw MgCI2. Thy and FUra degradation was assayed by a radiochemical method involving production of radioactive catabolites from [14C]Thy and RESULTS [14C]FUra. The assay mixture contained 20 Â¿Â¿Mofradioactive Thy or Pyrimidine Catabolism by Liver Extracts. The in vitro catab- 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 olism of BVUra was compared with that of Thy and FUra. For reaction was stopped by 30% perchloric acid; precipitated proteins were these experiments, liver extracts were used, since the liver eliminated by centrifugation. After neutralization, 10 /*! of the supernatant appears to be the major site for pyrimidine catabolism in vivo (7- were spotted onto Merck cellulose (for Thy) or silica gel (for FUra)-coated 9). When Thy was incubated with crude liver extract in the plates. Ascending chromatography was carried out using as eluents a presence of NADPH (under the conditions described in "Materials mixture of fert-butyl alcohol:methylethylketone:water:ammonium hydrox and Methods"), it was rapidly transformed to 0-ureidoisobutyric ide (40:30:30:10) (26) for cellulose plates or a mixture of chloro- acid and 0-aminoisobutyric acid with an initial velocity of 35 nmol/ form:methanol:acetic acid (30:10:5) (27) for silica gel plates. The plates h/mg protein. No H2Thy was detected, which suggests that the were scanned with a thin layer radiochromatogram scanner to locate and integrate the radioactive products. Inhibition studies of pyrimidine reductive conversion of Thy to H2Thy is the limiting step in the degradation by BVUra were performed under the same conditions but catabolism of Thy. When assayed under the same conditions, with different concentrations of inhibitor as described in the legends to FUra was degraded twice as fast as Thy (70 nmol/h/mg protein), the charts. From the crude liver extracts, H2Thy dehydrogenase was a-fluoro-ÃŸ-ureidopropionic acid and a-fluoro-/3-alanine were de partially purified by acid precipitation, ammonium sulfate fractionation, tected in the incubation medium while 5-fluorodihydrouracil was and DEAE-cellulose column chromatography (12); this last step enabled not. When Thy or FUra was incubated with H2Thy dehydrogen us to obtain part of H2Thy dehydrogenase free from the two subsequent ase separated from dihydropyrimidine cyclohydrolase and N- enzymes of the reductive catabolic pathway. carbamoyl-ÃŸ-alanine aminohydrolase by DEAE-cellulose chro Pharmacokinetics in the Rat. Nucleosides and bases were adminis matography, only the formation of H2Thy or 5-fluorodihydrouracil tered i.p. to adult male Wistar rats weighing approximately 400-500 g. could be demonstrated. In contrast with Thy or FUra, BVUra The schedule or administration is described in the legends to the charts. was not degraded when incubated with either crude liver extract The methods for plasma sample preparation and nucleoside and base quantitation by HPLC have been described previously (22). AUC (Â¿iMXh) or partially purified H2Thy dehydrogenase. for the plasma Thy and FUra concentrations were determined by the In Vitro Inhibition of Pyrimidine Degradation by BVUra. trapezoidal rule from time 0 to the last data point by first order extrapo BVUra not only was not a substrate of H2Thy dehydrogenase lation to infinite time. The same procedure was followed for the urine but acted as an inhibitor of this enzyme. Indeed, when liver Thy and FUra concentrations. extract was preincubated at 37Â°Cwith BVUra, the degradation In Vivo Inhibition of Pyrimidine Degradation by BVUra. BVUra (200 of both Thy and FUra was significantly reduced; the rate of Â¿imol/kg)was injected Â¡.p.in rats 1 h before they were sacrificed. The degradation of both Thy and FUra decreased as a function of livers were excised and 105,000 x g supernatants were prepared and the BVUra concentration in the incubation medium (Fig. 2). A dialyzed as described above. H2Thy dehydrogenase activity was assayed 50% inhibition of the degradation of 20 UM Thy or FUra was in these supernatants. In vivo inhibition of H2Thy dehydrogenase by obtained at a BVUra concentration of 2-3 /UM.An identical 50% BVUra was followed by measuring plasma concentrations of Thy or FUra inhibitory dose was obtained for BVUra if partially purified H2Thy- in rats pretreated with BVUra or BVdUrd. Potentiation of FUra Effects in Leukemic Mice. P388 cells were dehydrogenase was used instead of crude liver extract. used for the in vivo studies on the antitumor activity of FUra. These cells The inhibition of pyrimidine degradation was markedly en were kindly provided by Dr. A. A. Ovejera of the National Cancer Institute hanced if BVUra was preincubated with the enzyme extract in the presence of NADPH (Table 1A). Indeed, when BVUra was added at the same time as FUra to the incubation medium, inhibition of FUra degradation occurred only at 23% of that obtained when BVUra had been preincubated with enzyme ex tract at 37Â°Cin presence of NADPH. When NADPH was omitted from the preincubation medium, inhibition was only 21% of the maximal inhibition indicating that in the absence of NADPH BVUra did not bind to the enzyme. Moreover, the binding of BVUra to the enzyme in the presence of NADPH was markedly Fig. 1. Structure of (E)-5-(2-bromovinyl)uracil. reduced when the preincubation was done at 4Â°C, since the

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was increased to 100 MM,it interfered with the binding of BVUra to the enzyme, since, under these conditions, the inhibition was only 7%. Thus, inhibition of H2Thy dehydrogenase occurred only when BVUra was preincubated with the enzyme extract in the presence of NADPH and was not reversed by extensive dialysis. These results are consistent with an irreversible inhibition of H2Thy dehydrogenase by BVUra, whereby BVUra competes with FUra for a common binding site. Kinetics analyses are in progress to confirm this hypothesis. BVdUrd did not show any inhibitory activity under conditions where it could not be degraded to BVUra, i.e., by pyrimidine nucleoside phosphorylases that may occasionally be present in H2Thy dehydrogenase preparations. In Vivo Clearance of Thy, FUra, and BVUra. When Thy or FUra were administered i.p. to rats at 200 ^mol/kg, Thy or FUra

BVUra (jjM) appeared in the plasma within a few minutes but were rapidly cleared from the bloodstream according to a first order process Fig. 2. Inhibitory effects of BVUra on the degradation of Thy and FUra by liver (Fig. 3) with a half-time of 22 and 14 min, respectively (Table 2). extract. The 105,000 x g fraction of liver extract was preincubated for 10 min at 37Â°Cwith BVUra at different concentrations and then assayed for Thy and FUra Similarly, when the pyrimidine deoxynucleosides dThd or FdUrd degradation as described in "Materials and Methods." The percentage of inhibition were administered Â¡.p.,they also appeared in the blood within a was determined from the velocities of degradation obtained in the presence and absence of BVUra. few minutes and were then rapidly eliminated from the circulation.

Table 1 Erteci of preincubation of the enzyme preparation with BVUra on the inhibition of FUra 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 cases NADPH and BVUra, were added to the reaction mixture. The enzyme activity was determined as previously described and the percentage of inhibition was determined in comparison to the control sample. In B, the enzyme extract was preincubated for 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 enzyme source 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..e 0 2 i. 60 2 i. 6 16a Hours following administration of either Thy or FUra 76 24" Fig. 3. Effects of BVUra on the clearance of Thy and FUra from the plasma of rats. Thy and FUra were administered i.p. at 200 Mmol/kg. Their plasma concentra Preincubation mixture tions 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. B BVUra (20 MM) NADPH (250 MM) The number of animals used are indicated in Table 2. o o Table 2 86 Ie Pharmacokinetics of Thy and FUra in the plasma of rats * NADPH added after preincubation. Each compound was administered i.p. at 200 Mmol/kg according to the sched ules described in Figs. 3 and 4. Mean Â±SD for f1/2and AUC were calculated from " Activity of control sample, 73 nmol/h/mg protein. c BVUra added after preincubation. the individual values obtained for each rat. The increases in f1/2or AUC are indicated in parentheses. " Preincubation at 4Â°Cinstead of 37Â°C. 8 With FUra at 100 MMin the preincubation medium. Thyfi/2 of AdministrationThy rats6 (min)22Â±5(x h)171 (MMx inhibition obtained with preincubation at 4Â°Cwas only 31 % of alone 1) Â±26(x 1) that obtained with preincubation at 37Â°C. BVUra +ThydThd 34 232 Â±47 (x10.5)14Â±3(X1) 15699.2)50Â±15(x Â±558 (x

These experiments indicated that BVUra might be irreversibly alone 1) linked to H2Thy-dehydrogenase. This hypothesis was verified by BVUra + dThd 53Plasma206 Â±47 (x 14.7) 1063 Â±321 (X21.3) dialyzing the enzyme extract after preincubation with 20 ^M BVdUrd + dThdNo. 199Â±23(x14.2)AUCÂ±135(X17.2)Plasma861 BVUra with or without NADPH or FUra, and assaying the re FUraFUra maining activity with FUra as substrate (Table 1B). When the (min)14Â±5(x h)66Â±13(x(MMx enzyme was preincubated at 37Â°Cwith BVUra in the absence alone 1) 1) of NADPH and then extensively dialyzed, no inhibitory activity BVUra +FUraFdUrd 43 745.3)21 Â±12 (x 5348.1)44Â±10(x Â±92 (x was observed. If NADPH was present in the preincubation alone Â±12(x 1) 1) medium before dialysis, the degradation of FUra was inhibited BVUra + FdUrd 33fi/z 82 Â±8 (x 3.9) 532Â±83(x 12.1) by 86%. If the concentration of FUra in the preincubation mixture BVdUrd + FdUrd7 77 Â±7 (x 3.7)AUC 480 Â±65 (x 10.9)

CANCER RESEARCH VOL. 46 MARCH 1986 1096

The corresponding bases resulting from the phosphorolysis of inhibitory effect of BVUra on pyrimidine degradation in vivo was the deoxynucleosides appeared in the plasma and were elimi first demonstrated by comparing the H2Thy dehydrogenase ac nated within 2-3 h (Fig. 4). Ura, 5-iodouracil, and 5-trifluoro- tivity in the dialyzed 105,000 x g fractions of livers removed methyluracil followed the same clearance pattern as Thy and from rats pretreated with BVUra (200 Mmol/kg) to the livers from FUra (data not shown). control rats. The degradation of FUra was inhibited by 97% in On the contrary, after i.p. administration at 200 Mmol/kg, liver extracts from rats pretreated with BVUra as compared to BVUra was eliminated from the plasma very slowly, so that it liver extracts from untreated rats. maintained a concentration of 50-70 MMfor at least 6 h. BVUra In vivo inhibition of the catabolism of Thy and FUra was further was then cleared from the plasma according to an apparent first demonstrated by monitoring the effect of BVUra on the plasma order reaction with a mean half-life of about 8 h. Consequently, concentrations and pharmacokinetic parameters of Thy and BVUra was still present in the plasma at a concentration of 5- FUra. When BVUra was administered at 200 Mmol/kg 1 h before 20 MM24 h after its administration. The mean AUC for BVUra in the i.p. injection of Thy or FUra at 200 Mmol/kg, plasma concen the plasma was about 1000 MMXh. Following i.v. administration trations, i* and AUC were increased (Fig. 3; Table 2). Similarly, at 200 Mmol/kg, BVUra reached a plasma concentration of 100 plasma concentrations, tvâ€žandAUC of Thy and FUra, generated MMat 20 min; the first order elimination gave a half-life of 7.5 h by the phosphorolytic cleavage of dThd and FdUrd, were in and an AUC of 1175 MMXh. When BVdUrd was administered i.p. creased when these nucleosides were adminstered 3 h after to rats it was rapidly transformed to BVUra upon the action of BVUra (Fig. 4; Table 2). When BVdUrd instead of BVUra was pyrimidine nucleoside phosphorylases (22). Consequently, administered, the plasma concentrations, f,/,, and AUC of Thy BVUra reached a plasma concentration of 70 MM 2-4 h after and FUra, generated from dThd and FdUrd, respectively, again BVdUrd administration and was then eliminated with a half-life were markedly increased (Fig. 4; Table 2). Other pyrimidine of 7.5 h; its mean AUC was 1075 MMXh. After i.v. administration bases, which are substrates of H2Thy dehydrogenase such as of BVdUrd, a maximal concentration of 60 MM BVUra was Ura, 5-iodouracil, and 5-trifluoromethyluracil also showed a reached within 2-4 h; the half-life of BVUra was about 7.1 h and marked increase in plasma, f,/,, and AUC following administration its plasma AUC was 782 MMXh. of BVUra or BVdUrd (data not presented). The peculiar behavior of BVUra most probably resulted from Inhibition of pyrimidine degradation was noted at 8, 18, 24, its resistance to the action of H2Thy dehydrogenase. Elimination and even 48 h after a single BVUra (200 Mmol/kg) administration, of BVUra from the circulation by the kidney was low. The amount although at 48 h BVUra was undetectable in the plasma. A dose of BVUra eliminated in the urine during the first 48 h following of BVUra, 25 Mmol/kg, was as efficient as a dose of 200 Mmol/ administration of BVUra accounted for only 1.5-10% of the kg, and BVUra, even at 3 Mmol/kg, reached 80% of its maximum amount of BVUra administered. The maximum renal excretion of inhibitory effect. At lower doses the inhibitory effect rapidly BVUra occurred between 4 and 24 h after its administration and declined. reached peak urinary concentration of 700 MM.After 48 h, at a In Vivo Enhancement of the Antileukemic Effects of FUra time when BVUra could not longer be detected in the plasma, by BVUra. Since BVUra protected FUra from degradation not low concentrations of BVUra were still present in the urine. only in vitro but also in vivo, BVUra might be expected to enhance HPLC analyses demonstrated the presence of BVdUrd and the antitumor activity of FUra. This turned out to be the case as (E)-5-(2-bromovinyl)uridine in the urine as identified by their re illustrated by the experiments shown in Fig. 5. When DBA/2 tention time and by the action of pyrimidine nucleoside phospho mice were inoculated i.p. with 1 million P388 leukemia cells, they rylases. Since these two products were not present in the plasma died within 10 days after tumor cell inoculation. When FUra was at the time they were detected in the urine, it could be assumed administered at either 2.5 or 5 mg/kg at days 1,3,5, and 7 after that they were formed by a pentosyl exchange reaction at the kidney level. in Vivo Inhibition of Pyrimidine Degradation by BVUra. The ii*l â€¢; ra 15

' r

o-1- 25 5 20 0 10 200 0 2 l> 0 2 I, FUra Img/kg! FdUrd /mg/kg) Hours following administration of cither dThdor FdUrd Fig. 5. Effect of a previous administrationof BVUraon the antileukemicactivity Fig. 4. Effects of BVUraand BVdUrd on the plasma clearanceof Thy and FUra of FUraand FdUrdin mice bearingP388leukemiacells. DBA/2 micewere inoculated generated from the cleavage of their corresponding deoxynucleosides in rats. i.p. with 1 million P388 leukemia cells at day 0. At days 1, 3, 5, and 7 after cell PlasmaThy and FUraconcentrations were determined at the indicatedtimes after inoculation,the mice were treated i.p. with varying doses of FUraor FdUrd,without i.p. administrationof dThd or FdUrd at 200 >imol/kg,without (O)or with a previous P) or with (H) administration of BVUra (200 ^mol/kg) 1 h before FUra or FdUrd. administration of BVUra, 200 Â¿imol/kg(â€¢)orBVdUrd (â€¢),3h before the injection For each treatment group the mean day of death Â±SD (bars) is presented. of dThd or FdUrd.Bars, one SO. Numbers in parentheses, number of mice in each group.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1986 American Association for Cancer Research. INHIBITION OF PYRIMIDINE DEGRADATION BY BROMOVINYLURACIL tumor cell inoculation, it increased the survival of mice to 118 alone at 200 Mffiol/kg had neither antitumor nor apparent toxic and 128%, respectively, as compared to the control group (Table effects (Table 3). 3). However, when BVUra (200 /Â¿mol/kg)was administered 1 h FdUrd at 10 mg/kg had a very slight antileukemic effect which before each FUra injection, the survival was further increased to was considerably increased (mean survival time, 159%) when 150 and 176%, respectively. With these FUra doses, survivals FdUrd administration was preceded by BVUra; FdUrd alone at of BVUra-treated mice were significantly increased in comparison 200 mg/kg was well tolerated and increased the mean survival to those of mice receiving only FUra (Table 4). At 20 mg/kg, FUra time of mice to 164% in comparison to the control group but alone increased the mean survival time of leukemic mice to became toxic (as demonstrated by a marked loss of weight) if 158%, and if this dose of FUra was preceded by BVUra at a BVUra had been injected previously; in this case the survival dose of 200 ^mol/kg, the life span decreased to that of the time was lower than for the control group (Fig. 5). The survival control mice. When compared to that of mice receiving FUra at time of mice pretreated with BVUra (as compared to that of mice 20 mg/kg alone, the survival of BVUra-pretreated mice receiving receiving FdUrd only) was considerably increased at low doses FUra at 20 mg/kg was considerably decreased (Table 4). Clearly, of FdUrd while it was significantly decreased at high doses of this treatment regimen was toxic for the mice, as particularly FdUrd (Table 4). demonstrated by a marked loss of weight. BVUra administered Almost identical effects were obtained if BVdUrd (200 u.mo\l kg) instead of BVUra was injected 90 min before the administra

Table 3 tion of FUra or FdUrd (Fig. 6; Table 4). The time interval of 90 Effects of FUra, FdUrd, and inhibitors ofpyrimidine degradation on the survival of min was chosen, since after 90 min BVdUrd was almost com mice bearing P388 leukemic cells pletely cleared from the circulation and replaced by BVUra. DBA/2 mice inoculated i.p. at day 0 with 1 million P388 cells received at days The enhancing effect of BVUra and BVdUrd on the antitumor 1,3.5, and 7 an i.p. administration of the compounds listed below. Control mice received only saline. Survival times are presented as the mean Â±SD and in activity and toxicity of FUra in DBA/2 mice could be attributed percentage as compared to the control group. The number of animals per group is to an inhibition of the degradation of FUra and hence an increase indicated in parentheses. in the therapeutically active concentrations of FUra. Indeed, the CompoundNoneFUra (days)9.7 ofcontrol100118a plasma concentrations of FUra in mice, as in rats, were consid Â±0.8(48)1 erably increased if the mice had received BVUra or BVdUrd before FUra (Table 5). (2.5 mg/kg) 1.4 0.4 (6) 128a FUra (5 mg/kg) 12.4 1.3(24) In Vivo Effects of Some Other Inhibitors. We have also 158a107" FUra (20mg/kg)FdUrd 15.31.5(24)10.4 examined the effects of in vivo pyrimidine degradation of some other inhibitors of H2Thy-dehydrogenase, i.e., DUra (16,18,19), (10 mg/kg) 1.0(12) 121a FdUrd (50 mg/kg) 11.7 1.3(12) 164Â°94A1056 FdUrdmg/kg)BVUra (200 15.9 Â±1.4(12)9.2

(200 >imol/kg) Â±0.9 (6) BVdUrd (200 /Â¿mol/kg) 10.2 Â±0.8 (6) 1Â¿M 129a DUra (200 /^mol/kg) 12.5 Â±2.9 (6) 135" â€¢o 15 IS DUra (50 ^mol/kg) 13.1 Â±0.8(6) 1016 EtUra (200 ^mol/kg) 9.8 Â±0.8 (6) 97o" 6-AThy (70 /imol/kg)Survival 9.4 Â±0.6 (6)% Significantly different (P < 0.001) from control. 6 Not significantly different (P > 0.05) from control. 10 10

Table 4 Influence of inhibitors of pyrimidine degradation on the effects of FUra and FdUrd on 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. C- 0- administration of a H2Thy dehydrogenase inhibitor. Control mice received FUra or 0 25 B 20 O 10 50 200 FdUrd only. Results are expressed as the percentage of survival compared to the fÃ¼rilmg/kgl FdUrd Img/kg) corresponding control group. Fig. 6. Effect of a previous adminstration of BVdUrd on the antileukemic activity Inhibitors of pyrimi (mg/kg)5100 of FUra or FdUrd in mice bearing P388 leukemia cells. The experimental conditions dine degradation are identical to those explained in the legend to Fig. 5, except that BVdUrd (200 Oimol/kg)None Aimol/kg) instead of BVUra was administered 90 min before FUra or FdUrd.

150.2a 137.4a 60.5a BVUra (200) 151.1a10100 141.1a 60.5a Tables BVdUrd (200) 86" 37.6a DUra (200) Influence of BVUra and BVdUrd on plasma concentrations of FUra in mice 141.1a 65.4a200100 DUra (50) Plasma concentrations of FUra were determined 20 min after i.p. administration 98.8" EtUra (200) of different doses of FUra to DBA/2 mice. BVUra or BVdUrd was administered i.p. 106.2"FdUrd 6-AThy(70)None at 200 nmd/kg 1 h before FUra. (mg/kg)50100 ofFUra concentrations of FUra (>IM)upon injection FUra (mg/kg)5 alone1 +FUra21 +FUra26(61.5) (13.4)a 148a 54a BVUra (200) 1020 2 38.5 54 165.5aFUra 79.3a20100 48.3a BVdUrd (200)2.5100 2.5 (59) 79 159(189) * Significantly different (P < 0.001) from control. 50Plasma 4BVdUrd 308BVUra 273 6 Not significantly different (P > 0.05) from control. Numbers in parentheses, rat plasma values.

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EtUra (14), and 6-AThy (28). When injected i.p. in rats at 200 dehydrogenase. This enzyme is a flavoprotein (12). It has been /jmol/kg, DUra increased by 15- and 23-fold, respectively, the demonstrated (see Ref. 30 for review) that several flavin-depend plasma half-life and AUC of FUra, while EtUra and 6-AThy only ent enzymes can be inhibited irreversibly by substrate analogues slightly increased the half-life and AUC of FUra. DUra, EtUra, and which contain a function which is converted to a highly reactive 6-AThy were also investigated for their enhancing effects on the group within the active site. This highly reactive group may then antileukemic activity of FUra in mice (Table 4). Only DUra at 50 interact with an essential protein residue or prosthetic group Â¿Ãinol/kgcauseda marked increase in the survival time of leu- before dissociation occurs, thereby causing irreversible inhibition. kemic mice over that achieved by FUra alone. Unlike BVUra, Although the bromovinyl is per se less reactive than the diazo DUra had itself some antileukemic activity (Table 3), and when group (31, 32), its reactivity may be enhanced considerably in used at 200 ^mol/kg in conjunction with FUra (20 mg/kg) it the presence of NADPH, since this cofactor is necessary during effected a dramatic decrease in the mean life span of the mice the preincubation period to ensure irreversible binding of BVUra (Table 4). to the enzyme. BVUra inhibits the degradation of Thy and FUra in vivo. When rats are pretreated with BVUra before the administration of Thy DISCUSSION or FUra, plasma AUC of Thy and FUra (Table 2) is considerably increased (9.2- and 8.1-fold, respectively). Under these condi The present results demonstrate that BVUra is more slowly tions, BVUra probably acts by blocking the H2Thy dehydrogen cleared from the bloodstream than the other pyrimidine bases ase since the activity of this enzyme is markedly suppressed in probably because it is not a substrate for the first enzyme of the liver extracts from rats pretreated with BVUra. Based on its reductive pathway for pyrimidine degradation, i.e., H2Thy dehy inhibitory effect on pyrimidine degradation BVUra may be ex drogenase. Indeed, BVUra is not degraded by liver extracts pected to increase the in vivo therapeutic activity of 5-substituted which are rich in H2Thy dehydrogenase (7-9) or by partially Ura analogues such as FUra. In the experimental model used in purified enzyme. In vivo, it takes 48 h before BVUra is entirely this study, BVUra enhanced the antitumor activity of FUra, so cleared from the bloodstream after it has been administered to that FUra, at 5 mg/kg, in combination with BVUra was more rats i.p. at 200 /Â¿mol/kg.The plasma AUC values for BVUra effective than FUra alone at 20 mg/kg (Fig. 5). The increased obtained after i.p. or i.v. adminstration of BVUra (1000 and 1175 therapeutic activity of FUra in leukemic mice with BVUra is MMXh, respectively) are clearly higher than those obtained for probably due to an inhibition of FUra degradation, since in mice, Thy (171 and 222 /Â¿wxh,respectively) or FUra (66 and 144 like in rats, FUra plasma concentrations are considerably in MMXh, respectively), indicating that BVUra is more slowly cleared creased following BVUra pretreatment; yet, an exact correlation from the bloodstream than Thy and FUra. between plasma FUra concentration and increase in survival time BVUra is only partially eliminated by renal clearance. Although could not be established (Table 5; Figs. 5 and 6). Further studies, high concentrations (up to 700 Â¿JM)ofBVUra were found in the i.e., determination of peritoneal fluid concentrations of FUra may urine within the first 24 h following administration of BVUra, be necessary to evaluate such a correlation. urinary BVUra did not represent more than 10% of the adminis FdUrd at 10 mg/kg, in the presence of BVUra, has approxi tered BVUra. Possibly, BVUra could be transformed in vivo to mately the same antitumor activity as FdUrd alone at 100 mg/ BVdUrd by pyrimidine nucleoside phosphorylases (22) then phos- kg. It has been previously demonstrated that BVUra does not phorylated by cellular kinases and incorporated into nucleic acids. influence the cleavage of pyrimidine deoxynucleosides to the However, BVdUrd is not a substrate for cellular cytosol dThd free pyrimidines (22); thus, the enhancing effect of BVUra on the kinase; it only is a good substrate both for the mitochondrial antitumor activity of FdUrd is probably due to an inhibition of dThd kinase and herpes simplex and varicella-zoster virus-en coded enzymes (29).5 Thus, in uninfected cells or tissues BVdUrd FUra degradation following the cleavage of FdUrd to FUra. In the rat, FdUrd is rapidly transformed to FUra, which attains lower can be phosphorylated only by mitochondrial dThd kinase. To plasma concentrations than those achieved by the administration what extent if any this phosphorylation contributes to the clear of FUra itself (Table 2). However, following a previous BVUra ance of BVUra from the circulation is not clear, however. There administration, the plasma FUra concentration achieved upon are various other metabolic pathways or other elimination routes equimolar administration of FUra and FdUrd are almost identical. (e.g., biliary, fecal) by which BVUra may be eliminated, and in This may explain why FUra at 5 mg/kg and FdUrd at 10 mg/kg addition, BVUra may be bound to and retained by plasma or have approximately the same antileukemic activity in the pres tissue proteins. ence of BVUra, whereas in the absence of BVUra, FdUrd doses The present results also demonstrate that BVUra acts as a of 100 mg/kg are needed to achieve the same antitumor effect powerful inhibitor of H2Thy dehydrogenase and thereby inhibits as FUra at 20 mg/kg. Plasma AUC values estimated for FUra the degradation of other pyrimidines that are substrates for this from the data of Table 2 (51, 103, 89, and 108 jiMXh upon enzyme both in vitro and in vivo. In vitro, BVUra reduces the administration of FUra, 20 mg/kg; FUra, 5 mg/kg plus BVUra; degradation of Thy and FUra (Fig. 2). The findings that (a) FdUrd, 100 mg/kg; and FdUrd, 10 mg/kg plus BVUra, respec preincubation with liver extract is required for the inhibitory effect tively) correspond to a maximal increase in survival, while AUC of BVUra on H2Thy dehydrogenase activity, (b) this inhibitory values obtained with FUra, 20 mg/kg plus BVUra and FdUrd, effect is not reversed by extensive dialysis, and (c) in vivo 100 mg/kg plus BVUra (410 and 1081 Â¿tMXh,respectively) cor inhibition of pyrimidine degradation is maintained after BVUra respond to a toxic effect. Thus, antitumor activity as well as has been completely cleared from the circulation, suggest that, toxicity of FdUrd and FUra seem to be determined by the AUC like DUra (16), BVUra may be an irreversible inhibitor of H2Thy of FUra. 5 Unpublished observations. The relatively weak activity of FdUrd in the P388 tumor model

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1986 American Association for Cancer Research. INHIBITION OF PYRIMIDINE DEGRADATION BY BROMOVINYLURACIL may Â¡npart be attributed to its own metabolism by the P388 of toxicity studies, and these have shown that BVdUrd does not cells, which are, in contrast to other tumor cells, rather poor in lead to untoward effects when administered i.p. to mice at 750- thymidine kinase activity (33). This would limit the phosphoryla- 1500 /imol/kg daily for 4 consecutive wk (42). Since BVdUrd is tion of FdUrd to 5-fluoro-2'-deoxyuridine-5'-monophosphate in rapidly transformed to BVUra in vivo, it can be surmised that these cells and consequently the inhibitory action of FdUrd on BVUra has no marked toxicity at doses up to 1500 /imol/kg/day. thymidylate synthetase (34) and its incorporation into DNA (35). In conclusion, in addition to its ability to serve as a precursor P388 cells also have little pyrimidine nucleoside phosphorylase for the generation of the potent and selective antiviral drug activity (33, 35) which means that they are virtually unable to BVdUrd in vivo (22), BVUra also appears to be a powerful inhibitor convert FUra to FdUrd or 5-fluorouridine. However, FUMP may of the degradation of pyrimidines (such as FUra) via the H2Thy be formed directly from FUra upon the action of pyrimidine dehydrogenase pathway, allowing the use of 4- to 8-fold lower phosphoribosyl transferase which appears to be quite active in doses of FUra to achieve an equivalent therapeutic effect in the P388 cells (33, 36). Moroever, Mulkins and Heidelberger (33) P388 leukemia mouse model. have demonstrated that P388 cells resistant to FUra have a reduced pyrimidine phosphoribosyl transferase activity in com ACKNOWLEDGMENTS parison to the wild-type cells. Thus, the inhibitory effects of FUra on P388 cells may be related to the conversion of FUra to FUMP The authors thank I. Belloc, F. Dupuch, M. Verstreken for their expert technical assistance and C. Callebaut, N. Grillon and L. Palmaerts for their valuable help in and the eventual incorporation of FUMP into RNA (37). According the preparation of the manuscript. to our observations BVUra could act by blocking the degradation of FUra, thereby making available greater amounts of FUra to be REFERENCES processed via pyrimidine phosphoribosyl transferase to FUMP, which may then give rise to an increased incorporation of FUra 1. Wastemack, C. Degradation of pyrimidines and pyrimidine analogsâ€”pathways and mutual influences. Pharmacol. Ther., 8: 629-651, 1980. into RNA of P388 cells. 2. Fink, K., Henderson, R. B., and Fink, R. M. /i-Aminoisobutyric acid in rat urine The inhibition of Thy and FUra degradation achieved in vivo following administration of pyrimidines. J. Biol. 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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1986 American Association for Cancer Research. Effect of (E)-5-(2-Bromovinyl)uracil on the Catabolism and Antitumor Activity of 5-Fluorouracil in Rats and Leukemic Mice

Claude Desgranges, Gabriel Razaka, Erik De Clercq, et al.

Cancer Res 1986;46:1094-1101.

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