[CANCER RESEARCH 44, 1852-1856, May 1984] Potentiation of 5-Fluoro-2'-deoxyuridine Antineoplastic Activity by the Uridine Phosphorylase Inhibitors Benzylacyclouridine and Benzyloxybenzylacyclouridine1

Ming Yu W. Chu,2 Fardos N. M. Naguib, Max H. lltzsch,3 Mahmoud H. el Kouni, Shih Hsi Chu, Sungman Cha, and Paul Calabresi

Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912 [M. Y. W. C., F. N. M. N., M. H. I., M. H. e. K., S. H. C., S. C., P. C.], and the Roger Williams General Hospital, Providence, Rhode Island 02908 [M. Y. W. C., P. C.]

ABSTRACT in contrast to normal tissues, many types of neoplasia are deficient or have a low activity of thymidine phosphorylase when At a nontoxic dose (50 ^M), the two potent uridine phospho- compared to that of uridine phosphorylase (13, 18, 21, 24, 28, rylase inhibitors, benzylacyclouridine and benzyloxybenzylacy- 30, 34-37). Therefore, it was proposed (27, 28) that uridine clouridine (BBAU), potentiated 5-fluoro-2'-deoxyuridine (FdUrd) phosphorylase inhibitors, when coadministered with FdUrd, growth inhibition of human pancreatic carcinoma (DAN) and, to might cause selective toxicity against the tumor but not the host a lesser extent, human lung carcinoma (LX-1) cells in culture. tissues, because the host tissues would retain the capacity to BBAU was more effective than benzylacyclouridine. BBAU (50 cleave FdUrd to FUra by thymidine phosphorylase. A series of UM)enhanced the cytocidal effect of FdUrd (1 t¿M,3hr) on DAN acylouridines was developed as specific inhibitors of uridine grown on soft agar from 75 to 88%. In antithymocyte serum- phosphorylase (27, 28). It was shown that these compounds immunosuppressed mice bearing DAN, the mean tumor weight significantly inhibited the cleavage of FdUrd in extracts of tumors in animals treated with FdUrd (50 mg/kg/day for 2 days) was which lack or have low activity of thymidine phosphorylase (28). 11% less than that of untreated controls. When BBAU (10 mg/ Among these compounds, BAU and BBAU are the most potent kg/day for 2 days) was coadministered, the mean tumor weight (27, 29). at Day 10 was 78% less than untreated controls, with no The present study demonstrates the enhancement of FdUrd apparent host toxicity, clearly demonstrating the potentiation of antineoplastic efficacy by BAU and BBAU on the human pan the antitumor effects of FdUrd by BBAU. The fact that DAN creatic carcinoma (DAN) and the human lung carcinoma (LX-1 ) responded better than LX-1 to benzylacyclouridine and BBAU cell lines in vitro and in vivo. It also indicates that these BAUs could be due, in part, to the lower relative activity of thymidine are more effective in cell lines having a lower ratio of thymidine phosphorylase to uridine phosphorylase in DAN compared to phosphorylase to uridine phosphorylase. A preliminary report LX-1. The activities of other enzymes involved in FdUrd metab has been presented (10). olism, thymidine kinase, uridine kinase, orotate phosphoribosyl- transferase, 5'-nucleotidase, and dihydrouracil dehydrogenase, MATERIALS AND METHODS did not differ between the two cell lines. Chemicals. BAD and BBAU were synthesized as described previously (27). RPMI-1640 medium, FBS, A/-2-hydroxyethyl-1-piperazine-N'-2- INTRODUCTION ethanesulfonic acid buffer, trypsin:EDTA, Hanks' balanced salt solution, The efficacy of the cancer chemotherapeutic agent FdUrd4 is and trypan blue stain (4%) were obtained from Grand Island Biological limited by its cleavage to the less effective base FUra (15, 26). Co. (Grand Island, NY); rabbit ATS was from M. A. Bioproducts, (Walk- For many years, there has been a great deal of interest in ersville, MD); and FdUrd was from Calbiochem-Behring (La Jolla, CA). developing inhibitors of FdUrd degradation (1-3,16, 19, 20, 27- All unlabeled pyrimidine compounds, EDTA, DTT, carbamyl-DL-alanine, - 0-alanine, ninhydrin, 5-phosphoribosyl-1 -pyrophosphate, and NADPH 29,33) which might enhance the cytotoxicity and/or the selective were obtained from Sigma Chemical Co. (St. Louis, MO); dimethylamino- toxicity of this drug. As reviewed recently (29), 2 enzymes are benzaldehyde was from Aldrich Chemicals (Milwaukee, Wl); [2-"C]thymi- responsible for the cleavage of FdUrd: thymidine phosphorylase dine, [2-14C]uridine, [6-14C]uracil, [6-14C]orotate, 5-[2-uC]FUra, and [2- (EC 2.4.2.4) and uridine phosphorylase (EC 2.4.2.3). However, 14C]FdUrd were from Moravek Biochemicals, Inc. (Brea, CA); Silica Gel G/UV2M polygram, CEL 300/UV254 cellulose, and 300 PEI/UV254 poly- 1This investigation was supported by USPHS Grants CA 25631, CA 34228, CA ethyleneimine-cellulose polygram TLC plates were from Brinkmann In 31650, CA 13943, and CA 20892 awarded by the National Cancer Institute, struments, Inc. (Westbury, NJ); ACS scintillant was from Amersham/ Department of Health and Human Services, and Grant CH 136 awarded by the Searle Corp. (Arlington Heights, IL); Omnifluor scintillant was from New American Cancer Society. 2To whom requests for reprints should be addressed, at the Roger Williams England Nuclear (Boston, MA); Grade 1 neutral aluminum oxide was Hospital. from Woelm (Waters and Associates, Framingham, MA); and bovine y- 3 Recipient of support from the Training Program in Cancer Research, USPHS globulin and dye reagent for protein estimation were from Bio-Rad Grant CA 02904, awarded by the National Cancer Institute, Department of Health Laboratories (Richmond, CA). and Human Services. ' The abbreviations used are: FdUrd, 5-fluoro-2'-deoxyuridine; ATS, antithymo Cell Culture. The in vitro experiments to test the effect of BAU and cyte serum; BAU, benzylacyclouridine or 5-benzyl-1-{2'-hydroxyethoxymethyl)- BBAU on the cytotoxic effect of FdUrd were performed using human uracil; BBAU, benzylacyclouridine or 5-(m-benzyloxybenzyl)-1-(2'-hydroxyethoxy- pancreatic (DAN) and lung (LX-1) carcinoma cells, established in this methylXiracil; FBS, fetal bovine serum; FUrd, 5-fluouridine; FUra, 5-fluorouracil; TLC, thin-layer chromatography; DTT, dithiothreitol; OMP, orotidine 5'-monophos- laboratory (7,9). Cells at early passages (passages 10 to 20) were stored phate; FUMP, 5-fluorouridine 5'-monophosphate. at -120°C in growth medium (RPMI-1640 medium:10% FBS:0.02 M N- 2-hydroxyethyl-1-piperazine-N'-2-ethanesulfonic acid buffer) containing Received November 7, 1983; accepted February 2, 1984.

1852 CANCER RESEARCH VOL. 44

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1984 American Association for Cancer Research. Potentiation of FdUrd Effects by BAU and BBAU

10% dimethyl sulfoxide. For the present experiments, cells were main Ci (pH 7.5):10% glycerol:2 rnw DTT] was used for preparations to tained at 37°in growth medium in plastic tissue culture flasks (25 sq estimate the activity of thymidine kinase (EC 2.7.1.75), undine kinase (EC 2.7.1.48), orotate phosphoribosyltransferase (EC 2.4.2.10), and 5'- cm). Cells were recovered from exponentially growing cultures by using a solution of trypsin (0.05 g/liter) and EDTA (0.02 mg/liter) in Hanks' nucleotidase (EC 3.1.3.5). Buffer B [20 HIM potassium phosphate (pH balanced salt solution (free of calcium, magnesium, and bicarbonate). 8):1 mM EDTA:1 mw mercaptoethanol] was used for preparations to Tightly capped flasks were incubated at 37°in a nonhumidified incubator. estimate the activity of uridine phosphorylase, thymidine phosphorylase, Each cell line was analyzed routinely and found to be free of Mycoplasma and dihydrouracil dehydrogenase (EC 1.2.99.1). The homogenate was first centrifuged at 30,000 x g for 30 min at 4°, contamination (6). Determination of the Concentration of Drug Giving 50% Inhibition and the supernatant fluid was recentrifuged at 105,000 x g for 1 hr at of Growth Compared to Non-Drug-treated Control Cells. Plastic tissue 4°.The 105,000 x g supernatant fluid (cytosol) was used for all enzyme culture tubes (Corning 25200) were seeded with 4.8-ml suspensions of assays except 5'-nucleotidase. The 105,000 x g pellet (microsomes) 2x10* cells/ml in growth medium and incubated at 37°.After a 24-hr derived from the homogenate in Buffer A was resuspended in the same incubation, 0.1 ml of BAD or BBAU (final concentration, 50 /JM) was buffer and used to determine the activity of 5'-nucleotidase. added to the cell cultures. FdUrd (0.1 ml) was added 5 min later for a Enzyme Assays. All assays were run under conditions where the final concentration of 0.1 or 1 UM. Sterile 0.9% NaCI solution (saline) (0.2 activity was linear with time and enzyme concentration. ml) was added to controls. All experiments were carried out in duplicate. Pyrimidine Nucleoside Phosphorylases. The activity was measured Control and drug-treated cells were counted by the trypan blue exclusion by following the formation of base from nucleoside. The assay mixture method, and the number of doublings was calculated after 72-hr incu contained 20 mw potassium phosphate (pH 8.0), 1 mM EDTA, 1 mM bation at 37°.The dose that produced 50% inhibition of cell growth was mercaptoethanol, substrate (1 mw uridine or FdUrd, 9 mCi/mmol, or 2 estimated from the plot of the number of cell doublings versus the HIM thymidine, 4.5 mCi/mmol), and 50 pi of enzyme preparation in a final logarithm of drug concentration. volume of a 100 n\. The incubation was carried out at 37°.At 40 min, Determination of Cell Survival in Soft Agar. Cell survival was deter 50-jil aliquots were withdrawn from the assay mixture and placed into 5 mined by the soft agar cloning technique (8). A solution of 0.11 g of ¡Aof 40% perchloric acid to stop the reaction. Proteins were removed Noble agar in 5 ml of double-distilled water was autoclaved for 15 min, by centrifugation, and 25 n\ of the supernatant fluid were neutralized cooled down to approximately 60°,and transferred to a 44°water bath with 50 n\ of 0.1 N KOH. After centrifugaron, 10 n\ of the supernatant for 10 min before use. Fifty ml of 20% FBS in RPMI-1640 medium were fluid were spotted on silica gel plates to separate the bases from added, and the pH was readjusted to 7.0 with sterile 0.1 N HCI. Three- nucleosides by TLC as described previously (28). mi aliquots were distributed into 15- x 125-mm Kimax sterile glass test Pyrimidine Nucleoside Kinases. Formation of nucleotides from their tubes in quadruplicate. Exponentially multiplying cells in plastic culture respective nucleosides was measured by the method of el Kouni and tubes (Corning 25200) containing 4.8 ml of growth medium were treated Cha (11). The assay mixture contained 0.1 M Tris-CI (pH 7.6), 0.25 mM with BBAU (0.1 ml) for 5 min before FdUrd (0.1 ml) was added. Final substrate (0.4 mCi/mmol), 2.5 mM ATP, 3 mM MgCI2, 20 mM NaF, 2 mM concentrations of BBAU and FdUrd were 50 and 1 */M, respectively. After DTT, and 25 to 50 p\ of enzyme extract in a final volume of 100 pi. a 3-hr incubation, cells were recovered by trypsinization and diluted to Reactions were started by the addition of extract and stopped by the the appropriate number (to give approximately 200 colonies/4 tubes) in addition of 100 n\ of 0.2 M EDTA (pH 7.6). Nucleosides were then RPMI-1640 medium containing 20% FBS and 0.12% agar. A 2-ml separated from nucleotides on Alumina columns (0.6 g in a Pasteur pipet suspension of cells was then added to each agar aliquot. The tubes with a glass wool plug). were mixed quickly and gently and kept upright in ice until the gel began S'-Nucleotidase. The activity was measured by determining P, liber to form. These tubes were placed in racks and incubated at 37°.After ated from UMP or dTMP (12). The assay mixture contained 0.1 M Tris- 14 and 21 days, colonies were counted twice with a Darkfield colony Ci (pH 8.5), 1 mM substrate (UMP or dTMP), 10 mM MgCI2, and 25 n\ of counter (American Optical). The degree of survival was expressed as enzyme preparation in a final volume of 0.5 ml. The reaction was started the fraction of cells that formed colonies (i.e., the number of colonies by the addition of enzyme, and after incubation for 30 min at 37°,it was divided by the number of cells added) relative to the control (100%). terminated by the addition of 0.5 ml of ice-cold trichloroacetic acid (10%). Animal Toxicity Studies. The toxicity of BBAU and FdUrd was tested The precipitated protein was removed by centrifugation, and the liberated in groups of 6 non-tumor-bearing female C57BL/6 x DBA/2 F, (hereafter phosphate was determined by the method of Chen et al. (5) by adding 1 called B6D2Fi) mice (18 to 20 g) (Simonsen Laboratories, Gilroy, CA). ml of color reagent (1 volume each of 6 N sulfuric acid, 2.5% ammonium Mice were treated with ATS on the schedule described below in the in molybdate, 10% ascorbic acid, and 2 volumes of water) and incubating vivo studies. On Days 1 and 2, the animals were given i.p. injections of at 45°for 30 min. The color density was determined at 820 nm with a BBAU at 0,10, 30, 50, 75, or 100 mg/kg/day in the presence or absence red filter in a Gilford Model 240 spectrophotometer. Controls were treated of FdUrd at 0, 50, 75, 100, 150, 200, or 250 mg/kg/day. Animals were in the same manner, except that the enzyme was added after addition observed daily for mortality and weight loss, as an indicator for drug of the acid. toxicity, over a 12-day period. Orotate Phosphoribosyltransferase. The activity was determined by In Vivo Chemotherapy Studies. The antitumor efficacy of FdUrd in measuring the formation of OMP, orotidine, UMP, and uridine from [6- vivo was determined in the ATS-immunosuppressed mouse xenograft 14C]orotate, or FUMP and FUrd from [2-14C]FUra. The assay mixture model (32). Female B6D2F, mice (18 to 20 g) were immunosuppressed contained 50 mM Tris-CI (pH 8.0), 1 mM DTT, 5% glycerol, 3.5 mM MgCI?, with s.c. injections of ATS on Days -1, 0, 1, 3, and 6 of a 12-day 2.5 mM 5-phosphoribosyl-1-pyrophosphate, 0.25 mM orotate (4 mCi/ experimental schedule. On Day 0, pancreatic carcinoma (DAN) cells were mmol) or 0.5 mM FUra (4 mCi/mmol), and 30 //I of cytosol in a final harvested from monolayer cultures and injected s.c. (2.5 x 106 cells/ volume of 150 ¡A.The reaction was started by addition of enzyme, and mouse) into the area just above the sternum. On Day 1, mice in the after incubation at 37°,it was terminated by boiling for 30 sec. Proteins drug-treated groups were given i.p. injections of FdUrd (50 mg/kg/day) were removed by centrifugation, and then 10 n\ of the supernatant fluid alone, BBAU (10 mg/kg/day) alone, or a combination of the 2 drugs for were mixed with 10 //I of standard solution (8 mw orotate: 10 mM 2 days. Tumors were excised and weighed on day 10. A decrease in orotidine:uridine:OMP:UMP) or (10 HIM FUra:FUrd:FUMP). When orotate body weight was used as the indication of host toxicity. was used as the substrate, the mixture was spotted on polyethylene- Preparation of Cell Extracts. Exponentially growing cells were har ¡mine-celluloseTLC plates. The plates were developed in distilled water vested from monolayer culture, then washed, and packed in ice-cold to remove salts, allowed to dry, and then redeveloped in 0.2 M LiCI. saline solution according to the method of Scholar and Calabresi (31). When FUra was the substrate, the mixture was spotted on silica gel The packed cells were homogenized at 4°in 3 volumes of the appropriate plates which were developed in chlorofornrmethanol (9:1, v/v). Spots buffer using a Polytron homogenizer (Brinkmann). Buffer A [0.1 M Tris- were identified by UV quenching. R, values for OMP, UMP, orotate,

MAY 1984 1853

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1984 American Association for Cancer Research. M. Y. W. Chu et al. orotidine, and uridine were 0.16,0.51, 0.62, 0.77, and 0.95, respectively, 88% (Table 2). and 0.02, 0.24, and 0.48 for FUMP, FUrd, and FUra, respectively. Spots Animal Toxicity. The toxicities of BBAU and FdUrd were were cut out, and the radioactivity was counted in 20 ml of Omnifluor. evaluated singly or in combination in non-tumor-bearing ATS Dihydrouracil Dehydrogenase. The activity was measured by follow mice. Neither BBAU alone (10, 30, 50, 75, or 100 mg/kg/day for ing the formation of dihydrouracil, carbamyl /i-alanine, and p-alanine from [6-'*C]uracil. The reaction mixture contained 10 HIM potassium phos 2 days) nor FdUrd as a single agent (50, 75, 100, 150, 200, or phate (pH 8), 25 UM [6-14C]uracil (56 mCi/mmol), 0.5 rriM mercaptoetha- 250 mg/kg/day for 2 days) produced any mortality, and the mean nol, 0.5 mm EDTA, 2 mw DTT, 5 mM MgCI2, 0.1 TDMNADPH, and 25 ^l weight change was not significantly different from that of the of cytosol in a final volume of 50 n\. The reaction was started by addition controls. BBAU at 30 or 50 mg/kg/day for 2 days in combinations of extract, incubated for 30 min at 37°,and then terminated by boiling with FdUrd at 50, 75, or 100 mg/kg/day for 2 days was not for 1 min. Proteins were removed by centrifugation; then, 5 (¿lof the toxic. However, a significant (p < 0.01) weight loss (1.76 g/ supernatant fluid were spotted on cellulose TLC plates which were mouse) was observed when the highest dose of BBAU tested prespotted with 5 v\ of standard solution (10 mM uracil-carbamyl ß- (100 mg/kg/day for 2 days) was coadministered with high doses alanine:f(-alanine:25 mw dihydrouracil). The plates were developed over of FdUrd (150 mg/kg/day for 2 days). night in the top phase of a mixture of n-butyl alcohol:H2O:ammonium In Vivo Chemotherapeutic Effect. Table 3 shows the effect hydroxide (90:45:15, v/v). Uracil was identified by UV quenching, and ß- of BBAU on the efficacy of FdUrd against xenografts of the alanine, by spraying with 0.2% ninhydrin in 95% ethanol. Dihydrouracil pancreatic carcinoma DAN in ATS-immunosuppressed mice. and carbamyl ß-alaninespots were identified by dying with 5% dimeth- ylaminobenzaldehyde in 50% ethanol:N HCI and heating after dihydrour FdUrd alone (50 mg/kg/day for 2 days) produced an insignificant acil was hydrolyzed to its hydantoin by spraying with 0.5 N KOH in 50% (p > 0.05) reduction (11%) in tumor weight. However, when the ethanol. Spots were cut out, and radioactivity was counted in 20 ml of same dose of FdUrd was coadministered with BBAU (10 mg/kg/ Omnifluor. day for 2 days), a significant (p < 0.05) reduction (78%) in tumor Protein Estimation. Determination of protein concentration was car weight was observed. FdUrd at the highest dose used (150 mg/ ried out by the Bradford method (4) using 7-globulin as a standard. kg/day for 2 days) reduced tumor weight by 72%. These results

RESULTS Table 1 Effects of BAU and BBAUon the antineoplasticactivity of FdUrd against human In Vitro Cytotoxicity. At a concentration of 50 fiM, neither BAU pancreatic carcinoma (DAN) and lung carcinoma (LX-1) in culture nor BBAU alone had any effect on cell growth (results not shown). Growth inhibition was calculated as the reduction in the number of cell doublings after 72-hr incubation as determined by the trypan blue exclusion method. Cells However, Chart 1 shows that BBAU at concentrations as low as were pretreated with BAU or BBAU for 5 min before the addition of FdUrd. In each 1 fiM significantly enhanced the inhibitory effect of 0.1 and 1 U.M experiment, the nontreated control cells reached at least 3 doublings. FdUrd on the pancreatic carcinoma (DAN) by 4.5- and 1.8-fold, inhibitionCell % of growth respectively. Table 1 shows that FdUrd alone at 0.1 and 1 UM lineDANLX-1FdUrd(MM)0.11.00.11.0None26 (50MM)70 (50MM)91 inhibited growth of DAN (i.e., the number of doublings in 72 hr) ±5.9a58 ±3.410019 ±2.810026 by 26 and 58%, respectively. However, when 50 U.MBAU or ±1.810 BBAU was added 5 min prior to the addition of FdUrd, the growth inhibition by 0.1 UM FdUrd increased significantly (p < 0.01) to ±3.174 ±2.782 ±1.2100 ±4.1+BAU ±5.1+BBAU 70 and 91%, respectively. Table 1 also shows that, in human " Mean ±S.E. lung carcinoma (LX-1 ), treatment with 50 /¿MBAUor BBAU also enhanced growth inhibition by 1 MMFdUrd from 74% to 82 and Table 2 100%, respectively. At the lower FdUrd concentration (0.1 U.M), Effecf ot BBAUon FdUrd cytocidal activity against pancreatic carcinoma (DAN) BAU and BBAU enhanced growth inhibition from 10% to 19 and cloned in soft agar Cellswere incubated with 50 MMBBAUfor 5 min before addition of 1 ¡MFdUrd. 26%, respectively. Similar results were obtained when the soft After incubationfor 3 hr, cells were recovered,washed twice, and grown in medium agar cloning technique was used. BBAU (50 /¿M)enhanced the containing20% FBS and 0.12% Noble agar. Colonieswere counted after 21 days. cytocidal effect of 1 UM FdUrd on pancreatic DAN from 75 to TreatmentControl ofsurvival100 ±2.7* FdUrd 25 ±1.4" 10* FdUrd + BBAU% 12 ±0.8" ' Mean ±S.E. 6 Significantly different at p < 0.001.

E IO5 N Table 3 Effect of BBAUin combination with FdUrd on tumor weight of humanpancreatic carcinoma (DAN)in ATS-immunosuppressedmice IO4 ofcontroltumor increase(g/mouse)1.80 wt"100 GroupControlBBAUFdUrdFdUrdBBAU2days)0105015010 io3 ±17"100 ±0.17"1.00 ±989 ±0.241.23 0.1 1.0 ±1828 ±0.341.42 ±5C22 FdUrd (jjM) ±0.211.20 ±10°wt Chart 1. The enhancement of FdUrd toxicity against human pancreatic carci ±0.41 + FdUrdDosage(mg/kg/dayfor50% noma (DAN)cells in culture by BBAU. Cells were incubated with 1 (/u (A) or 10 «IM (•)BBAUfor 5 min before the addition of FdUrd.Controls were treated with FdUrd aTumors were excised and weighed at Day 10. alone (•).Cellsurvival was determined by the trypan blue exclusion method after 6 Mean ±S.E.from at least 5 animals in each group. 72-hr incubation. c Significantlydifferent at p < 0.05 from control value and from one another.

1854 CANCER RESEARCH VOL. 44

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1984 American Association for Cancer Research. Potentiation of FdUrd Effects by BAU and BBAU demonstrated that BBAU enhanced the antitumor effect of FdUrd the cleavage of FdUrd by 18.1 and 12.8% in DAN and LX-1, to the level achieved by a 3-fold-higher dose of FdUrd with no respectively. These figures represent the contribution of uridine apparent host toxicity. phosphorylase to the cleavage of FdUrd in each cell type. The Activities of the Enzymes Involved in FdUrd Metabolism. In results in Table 5 also indicate that the ratios of thymidine to an attempt to determine the basis for the differences in the uridine and FdUrd to uridine cleavage were both approximately therapeutic response to FdUrd and its potentiation by BBAU (or 2.6-fold higher in LX-1 than in DAN. BAU) in DAN and LX-1, we compared the activities of various enzymes known to be involved, directly or indirectly, in the DISCUSSION metabolism of FdUrd and FUra. These enzymes are uridine phosphorylase, thymidine phosphorylase, thymidine kinase, uri The present results demonstrate that the in vitro and in vivo dine kinase, 5'-nucleotidase, orotate phosphoribosyltransferase, antineoplastic efficacy of FdUrd can be significantly potentiated and dihydrouracil dehydrogenase. The results shown in Table 4 by the uridine phosphorylase inhibitors BAU and BBAU. The fact indicate that there were no significant differences (p > 0.05) in that BBAU is more efficacious than BAU correlates well with the the activities of any of these enzymes between DAN and LX-1. K, values of 32 and 98 nw estimated for BBAU and BAU as However, the ratio of thymidine phosphorylase to uridine phos inhibitors of uridine phosphorylase from sarcoma (S-180) cells phorylase between the 2 cell lines was significantly different (p (27). The enhancement by BAU or BBAU was particularly evident < 0.001). This ratio was calculated from each cell extract; with the human pancreatic carcinoma DAN. Although BAU or therefore, it should not be subject to variations between different BBAU alone (50 ^M) did not affect the growth of DAN in culture, batches of cells. Futhermore, Niedzwicki ef al. (28) have dem they enhanced the toxic effects of FdUrd (0.1 /¿M)from26 to 70 onstrated that this ratio is well correlated with the relative im and 91%, respectively (Table 1). BBAU (10 mg/kg/day for 2 portance of thymidine phosphorylase and uridine phosphorylase days) also enhanced the efficacy of FdUrd (50 mg/kg/day for 2 in the cleavage of FdUrd. days) against xenografts of DAN in ATS-immunosuppressed Table 5 shows the effect of BBAU on nucleoside phosphoryl mice as indicated by a reduction in the tumor weight from 11 to ase activity towards thymidine or FdUrd relative to that of unin 78%. To elicit a comparable effect in the absence of BBAU, a 3- hibited uridine phosphorylase activity. BBAU (25 U.M)inhibited fold-higher dose of FdUrd (150 mg/kg/day for 2 days) was uridine phosphorylase activity almost completely in both DAN required (Table 3). and LX-1. On the other hand, the cleavage of thymidine was not Although uridine phosphorylase is capable of cleaving FdUrd affected by BBAU, as reported previously (27). BBAU inhibited (Refs. 1, 2, 16, 17, 28, 29, and 34, and the present results), thymidine phosphorylase is the major enzyme reponsible for Table 4 FdUrd cleavage in tissues which contain both enzymes (Refs. 28 Activities of enzymes involved in FdUrd metabolism in human pancreatic and 34, and the present results). The present results confirm the carcinoma (DAN) and lung carcinoma (LX-1) previous findings (27) that BAU and BBAU inhibit uridine phos Activity (nmol/min/mg protein) phorylase but not thymidine phosphorylase. In the presence of EnzymeThymidine BBAU, only thymidine phosphorylase should be responsible for phosphorylaseUridine ±0.141s1 +0.1680.979 FdUrd cleavage. Therefore, the fact that the cleavage of FdUrd phosphorylaseThymidine .294 ±0.9040.036 ±0.4060.047 was approximately twice that of thymidine in the presence of kinaseUridine 41.133±0.01 +0.0111.760 kinasedTMP ±0.083.788 ±1.123.371 BBAU (Table 5) indicates that FdUrd is a better substrate than 5'-nucleotidaseUMP ±0.9013.831 ±0.725.39 thymidine for thymidine phosphorylase in these cell lines. In 5'-nucleotidaseOrotate ±1.9180.512 ±1.590.472 contrast to its lack of effect on thymidine cleavage, BBAU phosphoribosyltransferaseFUra +0.1260.424 ±0.0530.369 phosphoribosyltransferaseDihydrouracil +0.1440.01 ±0.0800.01 inhibited FdUrd cleavage by 18.1 and 12.8% in DAN and LX-1, dehydrogenaseThymidine 6±0.0010.140 4±0.0110.361 respectively (Table 5), which represent the contribution of uridine phosphorylase:uridine phos ±0.021"LX-10.356 ±0.072* phorylaseDAN0.186 phosphorylase activity towards the cleavage of FdUrd in the a Mean ±S.D. respective cell types. The activities of uridine phosphorylase and 6 Difference between the ratio (+ S.E.) for DAN and LX-1 was significant (p < thymidine phosphorylase did not differ significantly between DAN 0.001). and LX-1 (Table 4). However, this apparent lack of difference is probably due to the relatively large variations in activities be Table 5 tween different batches of cells, since the ratios of thymidine to Effect of BBAU on relative phosphorylase activity in human pancreatic carcinoma uridine and FdUrd to uridine cleavage were approximately 2.6- (DAN) and lung carcinoma (LX-1) fold higher in LX-1 than in DAN (Table 5). Relative activity is the ratio of uridine, thymidine, or FdUrd cleavage in the The differences in the metabolism of FdUrd between the 2 cell presence or absence of BBAU (25 UM) relative to that of uridine cleavage in the absence of BBAU. Specific activities of uridine phosphorylase in DAN and LX-1 lines DAN and LX-1 may be summarized as follows: (a) the ratio were 1.294 and 0.979 nmol/min/mg protein, respectively. of thymidine phosphorylase to uridine phosphorylase was ap- AdditionCell of inhibi proxmiately 2.6-fold higher in LX-1 than in DAN; (b) the rate of tion by FdUrd cleavage was 2.6-fold higher in LX-1 than in DAN; (c) the lineDANLX-1* BBAU97 ±0.23a extent of uridine phosphorylase participation in FdUrd catabolism was lower in LX-1 (12.8%) than in DAN (18.1%); and (d) the Thymidine 0.14 + 0.02 0.14 + 0.02 018.196 FdUrdUridine 0.33 ±0.031.00 0.27 ±0.030.04 degree of potentiation by BBAU is less in LX-1 than in DAN (Table 1). These findings, coupled with the fact that FdUrd was ±0.03 Thymidine 0.36 ±0.07 0.39 ±0.03 012.8 less toxic to LX-1 than to DAN (Table 1), support the original FdUrdNone1.00 0.86 + 0.09BBAU0.030.75 ±0.06% hypothesis (27, 28) that uridine phosphorylase inhibitors poten S.E.MAYMean + tiate the cytotoxic effects of pyrimidine nucleoside analogues

1984SubstrateUridine 1855

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such as FdUrd in those cells having little or no thymidine phos- 393, 1979. phorylase. 14. Handschumacher,R. E., Miller, A. M., and Levy, E. J. Tissue selective effects of benzylacyclouridineon the utilizationof circulatinguridine.Proc. Am. Assoc. Although the present results are consistent with the original Cancer Res., 24: 300, 1983. 15. Heidelberger,C., Griesbach, L., Cruz, O., Schnitzer, R. J., and Grunberg, E. speculations (27, 28), there are a few important factors, other Fluorinated pyrimidines. VI. Effects of 5-fluorouridine and 5-fluoro-2'-deoxy- than the ratio of thymidine phosphorylase to uridine phosphoryl- uridine on transplanted tumors. Proc. Soc. Exp. Biol. Med.. 97: 470-475, ase, which must be considered when evaluating the usefulness 1958. 16. Kelley, J. L., and Baker, B. R. Irreversibleenzyme inhibitors. 202. Candidate of BAU and BBAU in chemotherapy. It has been shown that BAD active-site-directedirreversible inhibitors of 5-fluoro-2'-deoxyuridine phospho and BBAU do not potentiate the effect of FdUrd in murine L5178Y rylase from Walker 256 rat tumor derived from 1-benzyl-5-(3-ethoxyben- leukemia cells5 but do inhibit nucleoside transport in L5178Y as zyl)uracil.J. Med. Chem., 25: 600-603,1982. well as human erythrocytes (22).5 Furthermore, it was demon 17. Krajewska, E., De Clercq, E., and Shugar, D. Nucleoside-catabolizingenzyme activities in primary rabbit kidney cells and human skin fibroblasts. Biochem. strated that BAU increases plasma uridine levels (14,23,25) and Pharmacol.,27: 1421-1426, 1978. the salvage of uridine by the various tissues to different degrees 18. Krenitsky, T. A., Barclay, M., and Jacquez, J. A. Specificity of mouse uridine phosphorylase.J. Biol. Chem. 239: 805-812, 1964. (14). Thus, it is apparent that the potential usefulness of BAU 19. Langen, P., and Etzold, G. 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Ming Yu W. Chu, Fardos N. M. Naguib, Max H. Iltzsch, et al.

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