Inhibitory Effects of Pyrimidine, Barbituric Acid and Pyridine Derivatives on 5-Fluorouracil Degradation in Rat Liver Extracts

Jpn. J. Cancer Res. (Gann), 78, 748-755 ; July, 1987

Kunihiko TATSUMI,Masakazu FUKUSHIMA,Tetsuhiko SHIRASAKAand Setsuro Funs Biwako Research Institute, Otsuka Pharmaceutical Co., Ltd, 11-1, 1-chome, Karasaki, Ohtsu, Shiga 520-01

The inhibitory effects of about 30 compounds, mainly pyrimidine and pyridine derivatives, on 5-fluorouracil degradation catalyzed by dihydrouracil dehydrogenase (DHU dehydrogenase) were investigated. The inhibitory activities of 5-substituted uracil derivatives decreased time- dependently during preincubation with liver extracts, indicating that these compounds are substrates of DHU dehydrogenase. During preincubation, 4,6-dihydroxypyrimidine derivatives were found to be converted to barbituric acid derivatives, which have stronger activities. The inhibitory activity of 2,4-dihydroxypyridine (3-deazauracil), which was stronger than that of uracil, did not change during preincubation, indicating that this compound is not a substrate but is an inhibitor of DHU dehydrogenase, and suggesting that 2,6-dihydroxypyridine (1-deazauracil) could be a potent inhibitor. In the light of these findings, we examined various derivatives of barbituric acid, 2,4-dihydroxypyridine and 2,6-dihydroxypyridine. Among these compounds, 3- cyano-2,6-dihydroxypyridine and 5-chloro-2,4-dihydroxypyridine were the strongest inhibitors with K values for DHU dehydrogenase of 2.3 x 10-'M and 3.6 X 10-'M, respectively.

Key words: 5-Fluorouracil - 5-Fluorouracil degradation - Pyrimidine - Barbituric acid - Dihydrouracil dehydrogenase

5 -Fluorouracil (5-FU) and uracil are pyrimidine degradation catalyzed by dihydro- catabolized by the same enzymes. The first uracil dehydrogenase, and they reported that step of their catabolism is reduction of the 5:6 this inhibitor increased the potency of the double bond of the pyrimidine ring. The first antitumor activity of 5-FU by decreasing its enzyme of this catabolic pathway, dihydro- degradation."' uracil (dihydrothymine) dehydrogenase (EC In the present work, we demonstrate that 5- 1.3.1.2), was identified as the rate-limiting substituted analogues of barbituric acid, 2,4- enzyme.'' Previous studies in this and other dihyroxypyridine, and 2,6-dihydroxypyridine laboratories have demonstrated that the deg- have stronger inhibitory activity than the in- radations of 5-FU and uracil are more rapid hibitors described above and that these in- in the liver than in other tissues.'4•6•" hibitors reduce the degradation of 5-FU by Various inhibitors of the degradation of 5- inhibiting dihydrouracil dehydrogenase activ- FU have been developed to enhance its thera- ity in vitro. These results suggest that the anti- peutic activity. These inhibitors include uracil tumor activity of 5-FU could be enhanced by analogues such as 5-ethyluracil, 5-iodouracil, co-administration with these potent inhibitors . 5-nitrouracil and 5 - bromouracil,5, 8-10) S- cyanouracil1' 1) and diazouracil,6,B, 11, 14) which MATERIALSAND METHODS all inhibit pyrimidine degradation in vitro or Chemicals and Animals [6-'H]5-Fluorouracil (5- in vivo.Most of these compounds are compet- FU) was obtained from the Japan Radioisotope itive inhibitors, since they are also substrates Association, Tokyo. 5-Fluorouracil, nicotinamide of dihydrouracil dehydrogenase .', 10,12, 15) adenine dinucleotide phosphate, reduced form We have reported increased antitumor ac- (NADPH), ribose 1-phosphate (Rib1P), deoxy- tivity by the use of a combination of a 5-FU ribose 1-phosphate (dRib1P) and 5-phospho- derivative and 2,4 - dihydroxypyridine in ribosyl-1-pyrophosphate (PPRibP) were pur- vivo.16)Recently, Desgranges et alLfound that chased from Sigma Chemical Co., USA. All other chemicalswere commercialproducts . Wistar-King (E)-5-(2-bromovinyl)uracil is not a sub- strain rats weighing about 180 g were purchased strate, but an inhibitor of the reductive step of from LABORIC Service Co. (Shiga). 748 Jpn. J. Cancer Res. (Gann) INHIBITORS OF 5-FU DEGRADATION

Assay of 5-FU Degradation Wistar-King strain used for assay of 5-FU phosphorylation. 5-FU is rats weighing 150-200 g were decapitated and the phosphorylated through three pathways as shown liver of each rat was rapidly perfused with saline in Fig 1. and removed. All subsequent procedures were For assay of phosphorylating activity through carried out at 4° . The livers were homogenized in 4 uracil ribosyltransferase and uridine kinase or ura- volumes of 0.25M sucrose containing 5mM 2- cil deoxyribosyltransferase and thymidine kinase, mercaptoethanol and 0.5mMethylenediaminetetra- the incubation mixture, in a final volume of 0.5 acetic acid (EDTA). The homogenate was cen- ml, contained Rib1P (2.0 umol) or dRib1P (2.0 trifuged at 105,000g for 60 min and the superna- umol), ATP (5.0 umol), MgC12 (2.5 umol), NaF tant was dialyzed overnight against 10mM potas- (5.0 umol), Tris-HCI (pH 8.0) (25,umol), ['H]5- sium phosphate buffer (pH 7.6). FU (1.0 iCi, 5 nmol), inhibitor solution (0.1 ml) Inhibition of 5-FU degradation was assayed by a and the enzyme solution (0.3 ml) in a final volume radiochemical method. The assay mixture, in a of 0.5 ml. For assay of pyrimidine phosphoribosyl- final volume of 1 ml, contained NADPH (0.1 transferase, the mixture contained PPRibP (2.0 ymol), adenosine triphosphate (ATP) (5.0 umol), umol), MgC12(2.5 umol), NaF (5.0 umol), Tris- nicotinamide (50 umol), sodium fluoride (NaF) HCI (pH 8.0) (25 umol), ['H]5-FU (1.0 uCi, 5 (25 umol), potassium phosphate (45 umol), ['H] 5- nmol), inhibitor solution (0.1 ml) and the enzyme FU (0.2,u Ci, 10 nmol), inhibitor solution (0.1 ml) solution (0.3 ml) in a final volume of 0.5 ml. and the liver extract (0.3 ml). The mixture was Mixtures were incubated at 37° for 10 min, mixed incubated for 5 min at 37° and the reaction was with 2M perchloric acid (100 ul) and centrifuged stopped by adding 0.1 ml of 40% trichloroacetic (3000 rpm, 10 min). Then, 100,u] of the superna- acid (TCA). The TCA was removed by extraction tant was added to 30 ul of 2M KOH solution and with ether, and 40 ul of the supernatant was 10 ul aliquots of the supernatant were subjected to spotted onto a thin layer chromatography (TLC) silica gel TLC with a mixture of chloroform, meth- plate (Merck silica gel 60 F254 precoated plates, 2 anol, and acetic acid (17:3:1, v/v). The spots of 5- X 20 cm, thickness 0.25 mm) and developed with a fluoronucleotides were scraped into vials. Subse- mixture of chloroform, methanol, and acetic acid quent procedures were the same as in the assay of (17:3:1, v/v). The spots of 2-fluoro-/3-alanine (F-/3- 5-FU degradation. Ala) and 2-fluoro-$-ureidopropionic acid (F-/.3- Assay of Thymidine Kinase The reaction mixture, UPA), which are 5-FU degradation products, were in a total volume of 0.5 ml, consisted of ATP (5.0 scraped into vials and extracted with 0.1 ml of 4M umol), MgC12(2.5 umol), Tris-HCl buffer (pH 8.0; HCI. The extracts were each mixed with 10 ml of 50umol), [6-'H]5-fluoro-2'-deoxyuridine (0.2pCi, scintillation fluid (ACS, Amersham) and the radio- 5 nmol), inhibitor solution (0.1 ml) and the activity was measured in an Aloka LSC-900 liquid enzyme solution (0.1 ml) prepared from sarcoma- scintillation spectrometer. IC,o values were deter- 180 cells. The mixture was incubated at 37° for 10 mined using this assay system. Three to five con- min and the reaction was stopped by adding 0.1 ml centrations of inhibitor producing between 30 and of 40% TCA. The TCA was removed and 40 ul of 70% inhibition of the reaction were used, and the the supernatant was applied to a TLC plate, and IC,, value was obtained from a plot of the percent developed with a mixture of chloroform, methanol, inhibition against the log of the concentration of and acetic acid (17:3:1, v/v). The spots of 5- inhibitor. fluoronucleotides were scraped into vials. Subse- Assay of 5-FU Phosphorylation The enzyme solu- quent procedures were the same as in the assay of tion was prepared from sarcoma-180 ascites tumor 5-FU degradation. cells. Tumor cells were homogenized in 4 volumes Isolation and Chemical Structure of the Active of 50mM Tris-HCl (pH 8.0), containing 5mM 2- Metabolite of 5-Chloro-4,6-dihydroxypyrimidine mercaptoethanol. The homogenate was centrifuged A sample of 190 mg of 5-chloro-4,6-dihydroxy- at 105,000g for 60 min, and the supernatant was pyrimidine was incubated with 1.5 liters of rat liver cytosol fraction for 24 hr at 37° and then 3.5 liters of ethanol was added. The mixture was allowed to stand for 2 hr, and then the ethanol-soluble fraction was evaporated at 40°, and the residue was chromatographed on a DEAE-Sepharose column developed with water containing an increasing concentration of HCI. The active fraction eluted with 0.1 N HCI was purified further by Sephadex LH-20 column chromatography with 8% butanol in water as the eluent, and the active metabolite was finally Fig. 1. Metabolism of 5-FU. obtained as a yellow powder (50 mg).

78(7) 1987 749 K. TATSUMI, ET AL. ypyrimidine with the Enzyme Extract on RESULTS 5-FU Degradation The inhibitors at concen- Effects of 5-Substituted Uracil and 4,6- trations of IO,uM were preincubated for 0, Dihydropyrimidine on 5-FU Degradation in 30 and 60 min with the liver extracts and Rat Liver Extracts The inhibitions of 5-FU cofactors at 37°, then 5-FU (10,tM) was degradation to 2 - fluoro - 3 - ureidopropionic added, and the inhibition of 5-FU degradation acid (F-l3-UPA) and 2-fluoroj3-alanine (F ,8- was determined as described above. As shown Ala) in rat liver extracts were measured as in Fig. 2, the inhibition of 5-FU degradation described above. As shown in Table I, all the was markedly enhanced by preincubation of compounds inhibited 5-FU degradation dose- the enzyme extract with 5-chloro-4,6-dihy- dependently, and of these compounds, thy- droxypyrimidine or 5-methyl-4,6-dihydroxy- mine was the strongest inhibitor. 5-Sub- pyrimidine, but significantly reduced by pre- stituted 4,6-dihydroxypyrimidine was less in- incubation with uracil or thymine. The inhibitory than 5-substituted uracil but 5-halo- hibitory activity of 2,4-dihydroxypyridine did genated derivatives tended to show higher not change during preincubation. These find- activities. ings prompted us to investigate why pre- Inhibitory Effects of Preincubations of Ura- incubation with 5 -chloro-4,6-dihydroxy- cil, Thymine and 5-Substituted 4,6-Dihydrox- pyrimidine enhanced the inhibition of 5-FU

Table I. Inhibitory Effects of 5-Substituted Uracil and 5-Substituted 4,6-Dihydroxypyrimidine on 5-FU Degradation in Rat Liver Extracts

Compounds were added at various concentrations to the reaction mixture for assay. Liver homogenate extract was used in the assay of 5-FU degradation. a) When 5-bromovinyluracil (BVUra) was added at the same time as 5-FU to the incubation medium, its IC,o was more than 100uMin our assay system. However, when preincubated with the enzyme extract in the presence of NADPH (IC,o: 3.5iM), it markedly enhanced inhibition of 5-FU degradation. This result is consistent with that of Desgranges et al."'

750 Jpn. J. Cancer Res. (Gann) INHIBITORS OF 5-FU DEGRADATION

Fig. 2. Effect of preincubation of the enzyme preparation on 5-FU degradation. Concentrations of IOaM inhibitors were incubated with liver homogenate extracts. After 0, 30 and 60 min, 5-FU was added to the incubation mixture and degradation of 5-FU was measured, as described under "Materials and Methods ." o, Uracil; O, thymine; A, 4,6-diydroxy-5-methylpyrimidine; •, 5-chloro- 4,6-dihydroxypyrimidine; O, 2,4-dihydroxypyridine.

Fig. 3. HPLC chromatogram of the reaction mixture before incubation (A) and after incubation for 60 min (B). Enzymatic conversion of 5-chloro-4,6-dihydroxypyrimidine to an active metabolite was analyzed by reverse-phase HPLC. 5-Chloro-4,6-dihydroxypyrimidine (1mM) was incubated with liver extract for 0, 10, 30 and 60 min. Then the supernatant obtained by filtration was injected into the column. Conditions for HPLC were as follows: Column, ULTRON N-C 18 L (4.6 ID X 150 mm); mobile phase, 10% MeOH-2mM TBA (pH 4.0); flow rate, 2 ml/min; detection at UV 260 nm. 78(7). 1987 751 K. TATSUMI, ET AL,

Table II. Spectral Data on 5-Chloro-4,6-dihvdroxvpvrimidine and Its Active Metabolite

The following spectometers were used: MS, JEOL JMS-EX-3000; IR, Shimadzu IR-435; UV, Beckman DU-8B; 'H-NMR, Hitachi R-90H.

degradation. The possible enzymatic conver- that the inhibitory activity of 2,4-dihydroxy- sion of 5-chloro-4,6-dihydroxypyridine to an pyridine did not change during preincubation, active metabolite was examined by high-pres- indicating that 2,4-dihydroxypyridine is not a sure liquid chromatography (HPLC). For substrate but is an inhibitor of dihydrouracil this, 1mM 5-chloro-4,6-dihydroxypyrimidine dehydrogenase. Therefore, we next tested the was incubated with liver extract and the reac- effects of derivatives of 2,4-dihydroxypyridine tion mixture was subjected to HPLC before and 2,6-dihydroxypyridine. and after incubation for 60 min (Fig. 3). As shown in Table IV, the inhibition of 5- During the incubation, a metabolite with a FU degradation was markedly enhanced by retention time of 13.4 min was formed. This both 2,4 -dihydroxypyridine and 2,6 -dihy- metabolite was isolated. As shown in Table II, its mass spectrum showed a molecular ion peak at 162 (m/z), indicating the introduc- Table III. Inhibitory Effects of 5-Substituted Bar- tion of one oxygen atom into 5-chloro-4,6- bituric Acid on 5-FU Degradation in Rat Liver dihydroxypyrimidine during its formation, Extracts The metabolite contained a pyrimidine ring, as shown by its UV spectrum (A"'Onm 267.5), and the presence of IR absorption bands at 1704 and 1610 cm-'. Furthermore its 'H- NMR spectrum lacked the signal of an aro- matic proton at 8.16 ppm (1H, s) seen in the spectrum of 5 -chloro- 2,4 -dihydroxypyrimidine. These results strongly suggested that this metabolite was 5-chlorobarbituric acid. Therefore, we next compared the inhibitions of 5-FU degradation by the metabolite and 5-chlorobarbituric acid. The two compounds showed similar patterns of inhibition of 5-FU degradation at various concentrations (data not shown). Thus the metabolite was identified as 5-chlorobarbituric acid. The inhibitory effects of 5-substituted barbituric acids on 5-FU degradation are shown in Table III; barbituric acid derivatives were more than 10 times stronger inhibitors of 5-FU degradation than 5-substituted 4,6-dihydroxypyrimidine or uracil derivatives. Inhibitory Effects of 2,4-Dihydroxypyridine and 2,6-Dihydroxypyridine Derivatives on 5- FU Degradation The results in Fig. 2 show

752 Jpn. J. Cancer Res. (Gann) INHIBITORS OF 5-FU DEGRADATION

Table IV. Inhibitory Effects of 2,4-Dihydroxy- droxypyridine derivatives, and in particular, 5-chloro- and 3-cyano-2,6-dihydroxypyridines pyridine and 2,6-Dihydroxypyridine Derivarives on 5-FU Degradation in Rat Liver Extracts (C1DP and CNDP, respectively) had very marked effects (more than 300 to 900 times those of thymine and uracil). Therefore, we next investigated whether these potent inhibitors of 5-FU degradation influenced the phosphorylation of 5-FU. Effects of Inhibitors of 5-FU Degradation on Phosphorylation of 5-FU in Sarcoma-180 Tumor Cell Extracts There are two pathways of phosphorylation of 5-FU to 5-fluorouridine- 5'-monophosphate (FUMP) (Fig. 1). One is catalyzed by pyrimidine phosphoribosyltransferase and the other by uracil ribosyltransferase and uridine kinase. As shown in Table V, these inhibitors did not affect the activity of pyrimidine phosphoribosyltransferase. 5- Chlorobarbituric acid and C1DP did not affect the phosphorylation catalyzed by uracil ribosyltransferase and uridine kinase, but CNDP at 10 and 100uM, inhibited this pathway 38.3 and 83.8%, respectively. This inhibition was much weaker than that of dihydrouracil dehydrogenase (ICs,, 0.06uM). The phosphorylation of 5-FU to 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) was not affected by 5-chlorobarbituric acid or C1DP. CNDP strongly inhibited the phosphorylation of 5-FU to HUMP, but even at 100uM,

Table V. Effects of Inhibitors of 5-FU Degradation on 5-FU Metabolism

NT=not tested. Various concentrations of the inhibitors of 5-FU degradation, 5-chlorobarbituric acid, 5- chloro-2,4-dihydroxypyridine and 3-cyano-2,6-dihydroxypyridine, were added to the reaction mixture for assay of 5-FU phosphorylation. ['H]5-FU (10iM) was incubated with sarcome-180 cell extracts in the presence of cofactor, as described in "Materials and Methods," and then the spots of F-nucleotides were analyzed by TLC.

78(7) 1987 753 K. TATSUMI, ET AL.

by the use of inhibitors of its degradation. Various 5-substituted uracils, diazouracil, and (E)-5-(2-bromovinyl)uracil were reported to inhibit the degradation of 5-FU by the pathway catalyzed by dihydrouracil dehydrogenase, which is the rate-limiting enzyme, and results showed that these inhibitors enhanced not only the antitumor activity of 5-FU but also its toxicity .1119)On the other hand, we found that when a masked form of 5-FU was given orally, its antitumor activity was enhanced by co-administration of uracil, uridine or thymidine without an appreciable increase in the toxicity, except in the case of thymine. Oral administration of tegafur and uracil (in a ratio of I to 4) increased the levels of 5-FU and HUMP in the tumor, with a greater increase in the 5-FU level in the tumor than in the blood and normal tissues,"' indicating that uracil inhibited the degradation of 5-FU but not its phosphorylation under these exper- imental conditions. In the present work, we studied the mode of action of 5-chloro-4,6- Fig. 4. Lineweaver-Burk plots of effects of in- dihydroxypyrimidine and found that during hibitors of 5-FU degradation. The initial velocity its preincubation with a liver homogenate it of dihydrouracil dehydrogenase was measured with was converted to 5-chlorobarbituric acid, various concentrations of 5-FU in the presence and which is a stronger inhibitor of 5-FU degrada- absence of inhibitiors. The reaction time was 5 min. tion than 4,6-dihydroxypyrimidine itself. This The K and K values, calculated from double re- finding led to us to test the inhibitory effects of ciprocal plots, were as follows: o , Without inhibitor derivatives of barbituric acid, 2,4-dihydroxy- (K, 3.6 X 10-'M); A, 5-chloro-2,4-dihydroxypyridine at 10-6M (K, 3.6x 10-'M); •, 3-cyano-2,6- pyridine and 2,6-dihydroxypyridine and we dihydroxypyridine at 10-6M (K, 2.3 X 10-'M) . found that 5 -chloro- 2,4 -dihydroxypyridine and 3-cyano-2,6-dihydroxypyridine were the strongest inhibitors. 5-FU is thought to have antitumor activity CNDP did not affect dThd kinase, indicating because it disturbs the maturation of rRNA that it inhibited uracil deoxyribosyltrans- and inhibits dTMP synthetase. We therefore ferase. There are several reports indicating tested the effects of these inhibitors on that uracil deoxyribosyltransferase does not phosphorylation of 5-FU. At low concentra- play an important role in animal tissues." tions, 5-chlorobarbituric acid and 5-chloro- K, Values of CIDP and CNDP on Dihydro- 2,4-dihydroxypyridine did not inhibit phos- uracil Dehydrogenase The K values of the phorylation of 5-FU. 3-Cyano-2,6-dihydroxy- inhibitors of 5-FU degradation were mea- pyridine inhibited uracil deoxyribosyltrans- sured with 5-FU as a substrate. As shown in ferase, but this enzyme is thought not to be Fig. 4, the results indicated that C1DP and important in animals.") Therefore, from these CNDP were competitive inhibitors of di- results, we conclude that these compounds hydrouracil dehydrogenease, which is in- inhibit 5-FU degradation and do not influence volved in 5-FU degradation, with K values of its phosphorylation, and that in combination 3.6 X 10-'M and 2.3 x 10-'M, respectively. with 5-FU or its masked form, these inhibitors DISCUSSION should be of therapeutic value. (ReceivedFeb. 4, 1987/AcceptedApril 22, 1987) There have been many studies on the en- hancement of the antitumor activity of 5-FU

754 Jpn. J. Cancer Res. (Gann) INHIBITORS OF 5-FU DEGRADATION

5-cyanouracil. Cancer Res., 30, 2937-2941 REFERENCES (1970). 12) Dorsett, M. T., Morse, P. A., Jr. and Gentry, 1) Canellakis, E. S. Pyrimidine metabolism. 1. G. A. Inhibition of rat dihydropyrimidine Enzymatic pathways to uracil and thymine dehydrogenase by 5-cyanouracil in vitro. degradation. J. Biol. Chem., 221, 315-322 Cancer Res., 29, 79-82 (1969). (1956). 13) Gentry, G. A., Morse, P. A., Jr. and Dorsett, 2) Frizson, P. Properties and assay of dihydro- M. T. In vivoinhibition of pyrimidine catab- uracil dehydrogenase of rat liver. J. Biol. olism by 5-cyanouracil. Cancer Res., 31, 909- Chem., 235, 719-725 (1960). 912 (1971). 3) Frizson, P. The relation between uracil 14) Ferdiandus, J. A. and Weber, G. Effect of 5- catabolizing enzymes and rate of rat liver diazouracil on the synthetic and catabolic regeneration. J. Biol. Chem., 237, 150-156 utilization of thymine in rat liver. Proc. Soc. (1962). Exp. Biol. Med., 139, 592-596 (1972). 4) Oueener, S. F., Morris. H. P. and Weber, G. 15) Shiotani, T. and Weber, G. Purification and Dihydrouracil dehydrogenase activity in properties of dihydrothymine dehydrogenase normal, differentiating and regenerating liver from rat liver. J. Biol. Chem., 256, 219-224 and hepatomas. Cancer Res., 31, 1004-1009 (1981). (1971). 16) Fujii, S. and Sakai, Y. Jpn. Patent No. 61- 5) Barrett, H. W., Munavalli, S. N. and New- 109719, 165-176 (1986). mark, P. Synthetic pyrimidines as inhibitors 17) Desgranges, C., Razaka, G., Clercq, E. D., of uracil and thymidine degradation by rat- Herdewijn P., Balzarini, J., Drouillet, F. and liver supernatant. Biochim. Biophys. Acta, 91, Bricaud, H. Effect of (E)-5-(2-bromovinyl)- 199-204 (1964). uracil on the catabolism and antitumor activ- 6) Ho, D. H. W., Haeberlen, R., Benjamin, R. ity of 5-fluorouracil in rats and leukemic S. and Bodey, G. P. Distribution of dihydro- mice. Cancer Res., 46, 1094-1101 (1986). uracil dehydrogenase in human tissues and 18) Wilkinson, D. S. and Crumley, J. Metabo- its inhibition by diazouracil. Proc. Am. Assoc. lism of 5-fluorouracil in sensitive and resis- Cancer Res., 22, 25 (1981). tant Novikoff hepatoma cells. J. Biol. Chem., 7) Ikenaka, K., Shirasaka, T., Kitano, S. and 252, 1051-1056 (1977). Fujii, S. Effect of uracil on metabolism of 5- 19) Cooper, G. M., Dunning, W. F. and Greer, fluorouracil in vitro. Gann, 70, 353-359 S. Role of catabolism in pyrimidine utiliza- (1979). tion for nucleic acid synthesis in vivo. Cancer 8) Goedde, H. W., Agarwal, D. P. and Eick- Res., 32, 390-397 (1972). hoff, K. Purification and properties of di- 20) Fujii, S., Ikenaka, K., Fukushima, M. and hydrouracil dehydrogenase from pig liver. Shirasaka, T. Effect of uracil and its deriva- Hoppe-Seyler's Z. Physiol. Chem., 351, 945- tives on antitumor activity of 5-fluorouracil 951 (1970). and 1- (2 - tetrahydrofuryl) - 5 - fluorouracil. 9) Newmark, P., Stephens, J. D. and Barrett, H. Gann, 69, 763-772 (1978). W. Substrate specificity of the dihydrouracil 21) Fujii, S., Kitano, S., Ikenaka, K. and dehydrogenase and uridine phosphorylase of Shirasaka, T. Studies on co-administration rat liver. Biochim. Biophys. Acta, 62, 414-416 of uracil or cytosine on antitumor activity of (1962). FT-207 or 5-FU derivatives. Jpn. J. Cancer 10) Sebesta, K., Bauerova, J. and Sormova, Z. Chemother., 6, 377-384 (1979). Inhibition of uracil and thymine degradation 22) Fujii, S., Kitano, S., Ikenaka, K. and by some 5-substituted uracil analogues. Bio- Shirasaka, T. Effect of coadministration of chim. Biophys. Acta, 50, 393-394 (1961). uracil or cytosine on the antitumor activity 11) Cooper, G. M. and Greer, S. Irreversible of clinical doses of 1-(2-tetrahydrofuryl)-5- inhibition of dehalogenation of 5-iodouracil fluorouracil and level of 5-fluorouracil in by 5-diazouracil and reversible inhibition by rodents. Gann, 70, 209-214 (1979).

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