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

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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 Unité8de l'Institut National de la Santéet de la Recherche Médicale,Avenue du Haut-Lévêque,33600 Ressac, France [C. D., G. R., F. D., H. B.¡,and Rega Institute for MédicalResearch, 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 ¿imol/kg)to rats, (£)-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 ß- 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 /¿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. (£)-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. Santéet de la Recherche Médicale,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, (£)-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 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1986 American Association for Cancer Research. INHIBITION OF PYRIMIDINE DEGRADATION BY BROMOVINYLURACIL 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 /¿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 ¿¿Mofradioactive Thy or Pyrimidine Catabolism by Liver Extracts. The in vitro catab- FUra, 250 I¿MNADPH, 35 rriM potassium phosphate, and liver extract (0.5 mg protein/ml of reaction mixture). After incubation at 37°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.
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