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In metastatic colorectal cancer the oral 5-fluorouracil (5FU) prodrug capecitabine is used with increasing frequency as an alternative to i.v. 5FU administration. The rate of conversion of capecitabine into 5'deoxy-5-fluorouridine has been related to tumor response, and 5FU catabolites have been associated with 5FU-related systemic toxicity. Here we demonstrate for the first time that capecitabine, its metabolites 5'deoxy-5-fluorocytidine and 5'deoxy-5-fluorouridine, and its catabolites 5-fluoro-ureido-propionic acid, alpha-fluoro-beta-alanine, and alpha-fluoro-beta-alanine-bile acid conjugate can be monitored in vivo by (19)fluorine magnetic resonance spectroscopy ((19)F MRS) in the liver of patients with metastatic colorectal cancer. Moreover, we demonstrate an improved signal-to-noise ratio and spectral resolution of the (19)F MRS spectra when measurements are performed at 3 T field strength as compared with measurements at the common clinical field strength of 1.5 T. We conclude that assessment of capecitabine metabolism in patients by (19)F MRS is a promising noninvasive tool for the prediction of its efficacy and toxicity, especially at the now currently available clinical field strength of 3 T.

5-(Benzyloxybenzyl)barbituric acid acyclonucleoside (BBBA) was recently synthesized as a potent and specific inhibitor of uridine phosphorylase (EC 2.4.2.3), the enzyme responsible for the catabolism of 5-fluoro-2'-deoxyuridine (FdUrd) in many types of tumors that are deficient or have little thymidine phosphorylase (EC 2.4.2.4) activity. The effect of BBBA on modulating the antitumor efficacy of FdUrd was evaluated in vitro, against the human colon carcinomas DLD-1 and HCT-15 grown in culture, and in vivo, against DLD-1 grown as xenografts in anti-thymocyte serum immunosuppressed mice. The concentrations of FdUrd that produced 50% growth inhibition after a 3-h exposure were 88 and 340 nM for HCT-15 and DLD-1, respectively. BBBA alone, at all concentrations tested, had no significant effect on the growth of DLD-1 and HCT-15 in culture. However, BBBA at 5, 10, 20, and 40 nM potentiated (P < 0.05) the cytotoxicity of FdUrd (340 nM; 3 h) against DLD-1 in culture by 20, 33, 55, and 63%, respectively. Similarly, BBBA at 10 and 20 nM potentiated the cytotoxicity of FdUrd (88 nM; 3 h) against HCT-15 in culture by 37 and 45%, respectively. In soft agar, BBBA (10 nM) also enhanced the cytocidal effect of FdUrd (10 and 32 nM) against DLD-1 by 41 and 55%, respectively, and against HCT-15 by 6 and 31%, respectively. Increasing BBBA dose to 20 nM enhanced further the FdUrd (10 and 32 nM) cytotoxicity against DLD-1 by 76 and 77%, respectively, and HCT-15 by 31 and 48%, respectively. BBBA also potentiated the chemotherapeutic efficacy of FdUrd in anti-thymocyte serum immunosuppressed mice bearing DLD-1 xenografts with no apparent host toxicity. At a low tumor burden (2.5 x 10(6) cells/mouse), 2 days treatment with FdUrd alone (50 mg/kg/day x 2) did not result in significant reduction in tumor volume. Coadministration of BBBA at 5 and 10 mg/kg/day x 2 did not potentiate the efficacy of FdUrd over that achieved by FdUrd alone, but it significantly reduced the tumor volume by 27 and 32%, respectively, when compared with untreated controls. FdUrd alone at 150 mg/kg/day x 2 reduced the tumor volume by 29%. This reduction in tumor volume was enhanced 1.8-fold by coadministration of BBBA (10 mg/kg/day x 2). At a higher tumor burden (5 x 10(6) cells/mouse) and 4 days treatment, BBBA at 10 and 30 mg/kg/day x 4 reduced further the tumor volume produced by FdUrd alone (200 mg/kg/day x 4) by 1.2- and 1.4-fold, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Enzyme inhibition studies on extracts from human liver, mouse liver, and human placenta indicate that there are considerable differences between human and murine hepatic uridine phosphorylases (UrdPase, EC 2.4.2.3) and thymidine phosphorylases (dThdPase, EC 2.4.2.4) with regard to their specificities and roles in the phosphorolysis of natural and 5-fluoropyrimidine nucleosides. To confirm further these differences between human and murine pyrimidine nucleoside phosphorylases, UrdPase and dThdPase were isolated from human liver, mouse liver, and human placenta using diethylaminoethyl-cellulose ion exchange chromatography. The pattern of elution from the column suggests that the hydrophobicity or charges on the human enzymes at pH 8 are different from those on their murine counterparts. The amount of each enzyme present differed between tissues and species. The apparent Km, Vmax, and efficiency of catalysis (Vmax/Km) values were determined for each enzyme using uridine, thymidine, deoxyuridine, 5-fluorouridine (FUrd), 5-fluoro-2'-deoxyuridine (FdUrd), and 5'-deoxy-5-fluorouridine (5'-dFUrd) as substrates. Kinetic parameters and inhibition studies were used to ascertain the binding affinity, substrate specificity, and contributions of UrdPase and dThdPase to the phosphorolysis of the various nucleosides in the 3 tissues. The roles of UrdPase and dThdPase in human liver were quite distinct from those of their counterparts from human placenta and mouse liver. In human liver, UrdPase appears to be highly specific to uridine. Human hepatic UrdPase contributes only 15% to the cleavage of FUrd and does not contribute to the cleavage of the deoxyribosides (thymidine, deoxyuridine, FdUrd, and 5'-dFUrd). In mouse liver, UrdPase has a broader specificity as it cleaves over 85% of FUrd, 15% of FdUrd, and 25% of 5'-dFUrd. On the other hand, human hepatic dThdPase has a broader specificity than murine hepatic dThdPase. Human hepatic dThdPase cleaves all nucleosides tested including the ribosides, uridine, and FUrd. Approximately 15% of uridine and 85% of FUrd phosphorolysis in human liver is carried out by dThdPase. This contrasts with the murine hepatic dThdPase, which is more specific to deoxyribosides, as it does not contribute to the phosphorolysis of uridine, and contributes only 15% toward the cleavage of FUrd. dThdPase is the principal enzyme responsible for the phosphorolysis of 5'-dFUrd in both human and murine livers. The specificities of UrdPase and dThdPase from human placenta resembled the enzymes from the murine liver more than those from human liver. Thus, it appears that the specificities of human hepatic pyrimidine nucleoside phosphorylases are distinct from those from extrahepatic tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

The pyrimidine acyclonucleoside benzyloxybenzyloxybenzylacyclouridine (BBBAU) showed growth inhibitory activity against the human colon cancer HCT-8 cell line with a 50% inhibitory concentration of 55 microM. Unlike its parent compounds, BBBAU was an extremely weak inhibitor of uridine phosphorylase. This acyclonucleoside analogue is an inhibitor of thymidylate synthase (TS) as determined by inhibition of [6-3H]-2'-deoxyuridine incorporation into DNA, inhibition of 3H release from [5-3H]-2'-deoxyuridine, and decrease in both the free and total TS 5'-fluoro-2'-deoxyuridine 5'-monophosphate binding sites. Kinetic analysis revealed that BBBAUMP, the monophosphate analogue of BBBAU, is a competitive inhibitor of purified human recombinant TS with a Ki of 8.0 microM. Nucleoside transport and uptake studies revealed that BBBAU (30 microM) inhibited the initial rate of transport and the total uptake of thymidine (25 microM). In contrast, while BBBAU (30 microM) inhibited the initial rate of transport of 5-fluoro-2'-deoxyuridine (FdUrd, 25 microM), its intracellular accumulation was increased. BBBAU (10 and 50 microM, respectively) potentiated FdUrd growth inhibition of HCT-8 cells and significantly enhanced the cytotoxic effects of FdUrd (0.3 and 1 microM, respectively) against HCT-8 cells using a clonogenic assay system. This combination resulted in additive inhibitory effects on TS activity resulting in greater depletion of dTTP pools. Moreover, the incorporation of radiolabeled FdUrd into the DNA fraction of HCT-8 cells was enhanced. The potential importance of this novel combination for human colon cancer chemotherapy is discussed.

The conjugation of antineoplastic drugs to monoclonal antibodies reactive with tumor associated antigens conveys selective cytotoxicity, overcoming the systemic toxicities caused by drugs during standard chemotherapy. 2'-Deoxy-5-fluorouridine, a more potent derivative of 5-fluorouracil, is an antimetabolite which exerts its cytotoxic action by inhibiting the enzyme thymidylate synthetase and as a result inhibits DNA synthesis. 2'-Deoxy-5-fluorouridine was successfully conjugated to anti-Ly-2.1 reactive with the murine thymoma ITT(1)75NS E3, I-1, and 250-30.6 reactive with human colon cancer cells using the active ester of 2'-deoxy-5-fluoro-3'-O-succinoyluridine (5FdUrdsucc). In vitro, 5Fd-Urdsucc-anti-Ly-2.1 was selectively cytotoxic against ITT(1)75NS E3 murine thymoma cells at nanomolar concentrations. The human colon carcinoma cell LIM1899 was inhibited by 5FdUrdsucc-I-1 conjugates in the range of 10(-7)-10(-8) M, as were Colo 205 cells by 5FdUrdsucc-250-30.6 conjugates. In vivo, 5FdUrdsucc conjugates were more effective than equivalent amounts of free 5FdUrdsucc. Against the ITT(1)75NS E3 murine thymoma, a single dose of 100 micrograms (5FdUrdsucc equivalents) 5FdUrdsucc-anti-Ly-2.1 resulted in 85% tumor inhibition compared to mean tumor size of control mice. Irrelevant 5FdUrdsucc conjugates failed to inhibit tumor growth. Multiple doses of 5FdUrdsucc-I-1 conjugate produced 50% growth inhibition of the moderately differentiated tumor LIM1899. In contrast, the human colon carcinoma Colo 205 was relatively resistant to free 5FdUrdsucc and 5FdUrdsucc-250-30.6 conjugates.

In vitro and in vivo data have indicated that tumor cells actively internalize the low density lipoprotein (LDL) from the circulation. In order to achieve a selective delivery of drugs to tumor cells via the LDL pathway, we have incorporated oleyl derivatives of methotrexate and floxuridine (FdUrd) into LDL particles. Three different incorporation procedures were studied: Method A, the dry film method; Method B, the transfer protein method; and Method C, the delipidation-reconstitution method. In all cases, 3H-labeled drug was incorporated into 125I-labeled LDL to yield double-labeled particles so that the behavior of both drug and carrier could be followed simultaneously. The last method led to the highest drug loading and it was possible to incorporate 50-70 molecules of dioleoyl-FdUrd per LDL particle as compared with about 18 molecules of drug when utilizing the transfer protein procedure. In vitro studies on the interaction of dioleoyl-FdUrd-LDL particles, obtained by the delipidation-reconstitution method, with the hepatocellular carcinoma cell line Hep G2, indicated that these reconstituted particles were equally effective in competing for LDL binding as native LDL. Moreover, drug delivery to Hep G2 cells occurred at the same rate as cellular association of the apolipoprotein B. In vivo studies on the fate of dioleoyl-FdUrd-LDL complexes in rats indicated that the serum decay was increased as compared with native LDL. The half-life of 6-9 min is, however, considerably prolonged as compared to the free drug (t1/2 less than 1 min). It is suggested that the 6-fold increased serum half-life of the drug-LDL complex accompanied by the possibly more specific tumor delivery may lead to an increased therapeutic effect.

[6RS]Leucovorin (5-formyltetrahydrofolate; 5-CHO-H4PteGlu) administered in different regimens in combination with 5-fluorouracil (FUra) has increased the response rates to FUra in patients with colon adenocarcinoma. Using preclinical models of human colon adenocarcinomas as xenografts in immune-deprived mice, the effect of the rate of administration of racemic [6RS]leucovorin on the concentration-time profile of reduced folates in plasma, size of intratumor pools of 5,10-methylenetetrahydrofolates (CH2-H4PteGlun) and tetrahydrofolates (H4PteGlun), and the distribution of their polyglutamate species have been examined. Bolus injection i.v., or 4-h or 24-h infusion of [6RS]leucovorin (500 mg/m2) yielded similar concentration profiles of the biologically active [6S] and inactive [6R] isomers of 5-CHO-H4-PteGlu and 5-methyltetrahydrofolate (5-CH3-H4PteGlu) in mouse plasma to those previously reported in humans, but with more rapid elimination half-lives (t1/2 = 11 to 16 min, 23 to 41 min, and 30 to 35 min, respectively). Thus, reduced folates remained elevated in plasma during the period of [6RS]leucovorin administration. In HxELC2 and HxGC3 tumors, pools of CH2-H4PteGlun and H4PteGlun were increased from 350% to 700% of control, but only during [6RS]leucovorin infusion. Intracellular levels subsequently declined rapidly, similar to the loss of reduced folates from plasma. Increasing the rate of [6RS]leucovorin delivery by decreasing the time for administration from a 24-h to a 4-h infusion did not further increase the intratumor pools of CH2-H4PteGlun and H4PteGlun, suggesting saturation in the cellular metabolism of [6RS]leucovorin. In HxGC3 tumors, CH2-H4PteGlu4-5 were elevated more rapidly than in line HxELC2, which accumulated predominantly a shorter chain length species following i.v. bolus injection. During the 4-h infusion schedule, di- and triglutamate species in particular accumulated in both tumors with no elevation in CH2-H4PteGlu5 until the infusion was discontinued, when this species increased as the shorter chain length forms were declining. However, during the 24-h infusion of [6RS]leucovorin, CH2-H4PteGlu3-5 were elevated in tumors. Since these species have been reported to increase the binding affinity of [6-3H]5-fluorodeoxyuridine monophosphate ([6-3H]FdUMP) to thymidylate synthase, and intratumor pools of CH2-H4PteGlun and H4PteGlun were elevated during the 24-h infusion of [6RS]leucovorin, this was considered to be the preferred schedule for administration.(ABSTRACT TRUNCATED AT 400 WORDS)

We have demonstrated previously the presence of 5-fluorouracil (FUra) residues in L1210 DNA. These findings have been extended to the MCF-7 human breast carcinoma cell line. Cesium sulfate gradient centrifugation has been used to separate the MCF-7 RNA and DNA fractions. Alkali and RNase digests have also been used to remove any possible RNA contaminating the DNA fraction. The purified DNA has been analyzed by high-pressure liquid chromatography following digestion to nucleotides and nucleosides. The results demonstrate that FUra residues are detectable in the DNA of these human breast carcinoma cells following exposure to either FUra of 5-fluorodeoxyuridine. Further, the extend of FUra incorporation in both MCF-7 RNA and DNA is similar with either fluorinated pyrimidine. We also demonstrate that the FUra incorporation in DNA from this human cell line can be enhanced by concurrent incubation with thymidine.

5-Fluorouracil and 5-fluorodeoxyuridine induce DNA lesions via two different mechanisms, one involving and the other not involving the incorporation of drug into DNA. The DNA lesions are detected by lysing cells in dilute alkali and then separating the DNA in agarose gel electrophoresis. We examine here the effect of dipyridamole, a nucleoside transport inhibitor, on the DNA lesions. We find that dipyridamole augments the levels of DNA fragmentation when the lesions are induced by the mechanism not involving the incorporation of drug. In parallel cytotoxicity is increased.

5'-Deoxy-5-fluorouridine (5'-dFUrd; Roche 21-9738) is a recently synthesized antineoplastic agent with therapeutic potential. The sensitivity of Ehrlich ascites tumor cells in CF-1 mice to 5'-dFUrd, as well as to 5-fluorouridine, was established. 5'-dFUrd was a more effective antitumor agent and was less toxic over a wide dosage range (50 to 400 mg/kg) than the other agents tested as measured by: (a) the ability to prevent gross development of inoculated tumor; (b) 45-day survival; and (c) weight change over the treatment period. With use of these sensitive tumor cells, the intracellular metabolism of 5'-dFUrd in vitro was investigated. Utilizing liquid chromatographic methodology for separation of acid-soluble metabolites, the only detectable metabolic products of 5'-dFUrd were FUra, 5-fluorouridine 5'-monophosphate, and 5-fluorouridine 5'-triphosphate. Novel metabolites of 5'-dFUrd were not detectable in the acid-soluble fraction or in plasma isolated from mice given [14C]5'-dFUrd. The formation of FUra appears to result from the action of nucleoside phosphorylase. 5'-dFUrd was shown to have a Km of 0.633 mM for this enzyme isolated from Ehrlich ascites tumor cells, an affinity similar to that for 5-fluoro-2'-deoxyuridine (Km, 0.278 mM) but much lower than that for 5-fluorouridine (Km, 0.049 mM). Incorporation of radiolabeled drug into the acid-insoluble fraction (representing greater than 95% incorporation into RNA) was also significant. 5-Fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) was not detectable as an acid-soluble metabolite. However, significant inhibition of thymidylate synthetase activity was detectable by 20 min in cells incubated with 30 microM 5'-dFUrd, suggesting that FdUMP was produced. The production of both 5-fluorouridine 5'-triphosphate and FdUMP appears dependent on the initial expansion of the FUra pool. This correlates with the inability of 5'-dFUrd to form nucleotide directly due to the absence of a 5'-hydroxyl group. It is concluded that the antineoplastic activity of 5'-dFUrd may be dependent on its enzymatic conversion of FUra. The basis for the possible increase in therapeutic index compared with other fluoropyrimidines may involve the rate and duration of the production of the biologically active nucleotides 5-fluorouridine 5'-triphosphate and FdUMP.

The pharmacokinetics of the new fluoropyrimidine 5'-deoxy-5-fluorouridine (5'-dFUrd) was investigated in twelve patients. The kinetics of the main metabolite 5-fluorouracil (5-FUra) was studied in eight patients and those of 5,6-dihydro-5-fluorouracil (5-FUraH2) in five patients. The patients participated in a Phase II study performed to investigate the response rate of 5'-dFUrd in advanced ovarian cancer. The pharmacokinetic data were compared with the clinical effects of the drug. The parent drug and 5-FUra were measured in both plasma and urine by high performance liquid chromatography. 5-FUraH2 concentrations in plasma were determined by capillary gas chromatography using electron capture detection and nitrogen-phosphorus specific detection. Several pharmacokinetic parameters such as elimination half-life, mean residence time, and steady state volumes of distribution are presented for 5'-dFUrd, 5-FUra, and 5-FUraH2. Two patients showed a partial response, four had stable disease, and five had progressive disease; one patient died due to myelotoxicity. The severity of the side effects correlated with the mean residence times of 5'-dFUrd and 5-FUra. Low pretreatment serum creatinine clearance (due to renal impairment) correlated with low renal excretion of 5'-dFUrd and a long mean residence time of 5'-dFUrd with the sum of observed toxicity. However, the extent of degradation of 5-FUra to 5-FUraH2 may also be related to the severity of the toxicity of 5'-dFUrd.

Isoelectric focusing and studies with 1-(2'-deoxy-beta-D-glucopyranosyl)thymine (GPT), a specific inhibitor of uridine phosphorylase activity, were used to determine the substrate specificities of mammalian pyrimidine nucleoside phosphorylases and their cleavage of 5-fluoro-2'-deoxyuridine (FdUrd). Isoelectric focusing profiles for the cytosol fractions from Ehrlich ascites cells and from Novikoff hepatoma cells each consisted essentially of one peak of nucleoside phosphorylase activity [isoelectric points (pl) 5.4 and 5.8, respectively] that cleaved both uridine and thymidine (dThd), as well as FdUrd. By contrast, cytosol fractions from HeLa (S3) cells, mouse liver, and normal human leukocytes each exhibited a major peak of activity (pl 4.6, 6.5, and 4.9, respectively) that cleaved only dThd and FdUrd, while mouse liver exhibited a second peak (pl 5.2) that cleaved primarily uridine. To distinguish clearly between (a) uridine phosphorylases that cleave primarily uridine and that are inhibited by GPT and (b)dThd phosphorylases that cleave only deoxynucleosides and that are not inhibited by GPT, we propose the term "uridine-deoxyuridine phosphorylases" to define those pyrimidine nucleoside phosphorylases that cleave both uridine and dThd and that are inhibited by GPT. On the basis of this definition and studies with GPT in nonfocused cytosol preparations, we conclude that FdUrd is cleaved to 5-fluorouracil by uridine-deoxyuridine phosphorylase activity in Ehrlich ascites cells and in Novikoff hepatoma cells, and by dThd phosphorylases in mouse liver, in normal human leukocytes, and in HeLa (S3) cells.

5'-Deoxy-5-fluorouridine (5'-dFUrd) is a new antineoplastic agent which possesses a higher therapeutic index in several experimental tumors compared to other fluoropyrimidines. During a Phase I trial, 5'-dFUrd, 1 to 15 g/sq m/week, was administered to patients as a 25- to 35-min i.v. infusion. Plasma kinetics and metabolism of 5'-dFUrd were investigated. The unmetabolized drug was measured by a high-performance liquid chromatography assay. 5-Fluorouracil and 5,6-dihydrofluorouracil, the two detected plasma metabolites, were quantitated by a gas chromatography-mass spectrometry methodology with a detection limit of 0.07 microM for both metabolites. The disposition of 5'-dFUrd in humans at therapeutic doses followed a nonlinear kinetic process. Plasma concentrations of 5-fluorouracil generated in vivo represented approximately 6% of 5'-dFUrd concentrations and the 5-fluorouracil half-life ranged from 8.8 to 27.1 min. High plasma values of 5,6-dihydrofluorouracil (14.5 to 30 microM) were observed in patients, indicating the importance of this pathway in humans.

The influx of 1.0 muM 5-fluoro[6-3H]deoxyuridine (5F[6-3H]dUrd) into L5178Y mouse leukemia cells followed a linear function with time from 2 to 10 min. Ammonium 5-bromodeoxyuridine 5'-methylphosphonate (BrdUrd-OPO2Me) inhibited the membrane transport of 5F[6-3H]dUrd into L5178Y cells. Influx of 5F[6-3H]dUrd into inhibited cells was observed from zero to 3 min; after 3 min the net rate of 5F[6-3H]dUrd uptake into the cells treated with 18 muM BrdUrd-OPO2Me was almost zero. The cellular uptake of 2'-deoxy[6-3H]uridine or 5-bromo[6-3H]deoxyuridine was inhibited by BrdUrd-OPO2Me. The L5178Y cells were grown for 96 hr in a medium that contained tritium-labeled BruDur-OPO2Me. An analysis of the labeled products in the growth medium showed that the ester linkage is not cleaved to separate the [3H]methylphosphonate group and the nucleoside moiety of BrdUrd-OPO2[3H]Me. The activity of thymidine kinase in a cell-free preparation from L5178Y cells was demonstrated. Although 37 muM 5-bromo-2'deoxyuridine produced an inhibition of approximately 45% in kinase activity, BrdUrd-OPO2Me had no effect on enzyme activity. The results indicate that BrdUrd-OPO2Me is an inhibitor of the cell membrane transport of the 5-fluoro and 5-bromo derivatives of 2'-deoxy[6-3H]uridine.

The effect of polyinosinic X polycytidylic acid [poly(I) X poly(C)] on the cell lethality produced by 5-fluorouracil [FUra] and 5-fluorouridine [FUrd] was examined in human colon carcinoma cell line HT-29. Pretreatment of cells with poly(I) X poly(C) as well as during exposure to FUra or FUrd resulted in antagonism of the cell lethality generated by the fluoropyrimidines. Antagonism of FUra cytotoxicity was also produced by the 2'-O-methylated analogue, polyinosinic X poly-2'-O-methylcytidylic acid, but not by the individual single-stranded polynucleotides or by the component mononucleotides, cytidine 5'-phosphate and inosine 5'-phosphate. In contrast, cytidine 5'-phosphate blocked the toxicity of FUrd. The antagonism by poly(I) X poly(C) of FUra and FUrd cytotoxicity was related to the inhibition of their metabolism to fluorouridine triphosphate and their incorporation into RNA and not to inhibition of the synthesis of RNA. Antibodies to leukocyte and fibroblast interferons did not reverse the antagonistic activity of poly(I) X poly(C). These results indicate that poly(I) X poly(C) may be interfering with the transport and/or initial metabolism of FUra and FUrd to fluorouridine monophosphate which is independent of the ability of the double-stranded RNA to induce interferon.

The influence of dose and hepatic blood flow on the elimination of 5-fluoro-2'-deoxyuridine (FdUrd) by the isolated perfused rat liver were investigated. FdUrd (1-20 mg; 4-81 mumol) was injected into the perfusion reservoir and serial samples were collected for chromatographic determination of plasma FdUrd and 5-fluorouracil concentrations. The decrease in FdUrd concentration from values above 100 microM was linear with time (apparent zero order); at concentrations below 30-40 microM the decline became exponential (apparent first order). Semilogarithmic plots of FdUrd concentration/dose versus time obtained with different doses were not superposable, indicating Michaelis-Menten elimination. At a perfusion rate of 20 ml/min, the apparent Vmax and Km for FdUrd disappearance were 14-19 nmol/ml/min and 161-194 microM, respectively. FdUrd clearance during first-order elimination was 8-11 ml/min. After FdUrd administration, 5-fluorouracil concentration reached 10-15% of the initial FdUrd concentration, then decreased with a half-life of 4-7 min. Fifty-four % of the dose of [2-14C]FdUrd was converted to 14CO2. At a dose of 20 mg, first-order clearance of FdUrd increased from 7 to 12 ml/min as hepatic flow increased from 10 to 30 ml/min. Less than 1% of the dose of [6-3H]FdUrd was incorporated into macromolecules. It was concluded that hepatic elimination of FdUrd is dependent on both dose and blood flow.

The use of a new methodology, 19F nuclear magnetic resonance, has allowed detection of all the fluorinated metabolites in the biofluids of patients treated with 5'-deoxy-5-fluorouridine (5'-dFUrd) injected i.v. at a dose of 10 g/m2 over 6 h. This technique, which requires no labeled drug, allows a direct study of the biological sample with no need for extraction or derivatization and a simultaneous identification and quantitation of all the different fluorinated metabolites. As well as the already known metabolites, unmetabolized 5'-dFUrd, 5-fluorouracil, and 5,6-dihydro-5-fluorouracil, the presence of alpha-fluoro-beta-ureidopropionic acid, alpha-fluoro-beta-alanine (FBAL), N-carboxy-alpha-fluoro-beta-alanine, and the fluoride anion F- is reported. The catabolic pathway proposed for 5'-dFUrd is analogous to that of 5-fluorouracil, completed with FBAL----F- step, and the plasmatic equilibrium of FBAL with N-carboxy-alpha-fluoro-beta-alanine, its N-carboxy derivative. The quantitative analysis of the different metabolites found in plasma and urine emphasizes the significance of the catabolic pathway. High concentrations of alpha-fluoro-beta ureidopropionic acid and FBAL are recovered in plasma from 3 h after the beginning of the perfusion to 1 h after its end. The global urinary excretion results show that there is a high excretion of 5'-dFUrd and metabolites. Unchanged 5'-dFUrd and FBAL are by far the major excretory products and are at nearly equal rates. The protocol followed in this study produces relatively low but persistent plasmatic concentrations of 5-fluorouracil throughout the perfusion.

Methods have been developed to assay several aspects of 5-fluoro-2'-deoxyuridine and 5-fluorouracil metabolism in tissue culture cells. These methods allow measurement of (a) intracellular levels of the covalent complex formed between 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP), 5,10-methylenetetrahydrofolate, and thymidylate synthetase; (b) incorporation of drug into RNA; and (c) analysis of drug metabolites. The methods were developed using radioactively labeled drugs, but they should be adaptable to studies using nonlabeled compounds. Sephadex G-25 chromatography or trichloroacetic acid precipitation were utilized for isolation of the macromolecular cell fraction; prior treatment of this fraction with RNase or heating at 65 degrees for 15 min resulted in selective removal of RNA or the thymidylate synthetase complex, respectively, from the precipitable fraction. Free intracellular drug metabolites present in the acid-soluble fraction were analyzed by high-pressure liquid chromatography. Following incubation of HTC cells with [6-3H]-5-fluoro-2'-deoxyuridine, a radioactive macromolecule was isolated and identified as a FdUMP-5,10-methylenetetrahydrofolate-thymidylate synthetase complex. Intracellular formation of this complex was shown to be dependent on the presence of the enzyme thymidine kinase. Dissociation of the complex in vivo was first order with a t1/2 of 6.2 hr; in contrast, a t1/2 of 2 hr was determined for dissociation of the complex in cytosol. Incubation of L1210 cells with [6-3H]-5-fluorouracil for 22 hr resulted in formation of 80 fmol of FdUMP-5,10-methylenetetrahydrofolate-thymidylate synthetase complex per 10(6) cells, as compared with 400 fmol of drug incorporated into RNA per 10(6) cells. Intracellular FdUMP could not be detected in the acid-soluble fraction of these cells unless the cells were first heated to dissociate the complex.

The intracellular metabolism of a new fluoropyrimidine, 5'-deoxy-5-fluorouridine (5'-dFUrd), was compared with the metabolism of 5-fluorouracil (FUra), 5-fluorouridine (FUrd), and 5-fluoro-2'-deoxyuridine (FdUrd) in freshly isolated bone marrow cells and Ehrlich ascites tumor cells. Following exposure to tumor cells, all four fluoropyrimidines were metabolized to identical products (i.e., FUra, 5-fluorouridine 5'-monophosphate, 5-fluorouridine 5'-diphosphate, 5-fluorouridine 5'-triphosphate, and 5-fluoro-2'-deoxyuridine 5'-monophosphate), all produced an incorporation of FUra into RNA (FUd greater than FUra greater than FdUrd greater than 5'-dFUrd), and all completely inhibited thymidylate synthetase activity by 1 hr. However, in bone marrow cells, very different patterns were observed. 5'-dFUrd accumulated in the cells, but there were no measurable metabolism, no incorporation of FUra into RNA, and no inhibition of thymidylate synthetase activity. In contrast, both FUra and FUrd were metabolized and produced an incorporation of FUra into RNA (2.7 pmol FUra per micrograms RNA and 4.8 pmol FUra per micrograms RNA at 2 hr, respectively) in bone marrow. Only a minor inhibition of thymidylate synthetase activity was detected. FdUrd also was metabolized by bone marrow cells, produced a low level of FUra incorporation into RNA (0.23 pmol FUra per micrograms RNA at 2 hr), and produced a complete inhibition of thymidylate synthetase activity. Since 5'-dFUrd is not directly cytotoxic itself, its superior therapeutic index compared to other fluoropyrimidines may largely reflect the selective activation of 5'-dFUrd by sensitive tumor cells as opposed to bone marrow cells, which can activate FUra, FUrd, and FdUrd.

The exposure of HL-60 human promyelocytic leukemia cells to 0.5 microM 5-fluoro-2'-[3H]deoxyuridine (FdUrd) for 16 hr resulted in the incorporation of 5.14 +/- 0.31 (S.D.) X 10(-7) mol FdUrd into DNA per mol of DNA nucleotide, which corresponds to 0.146 +/- 0.082 pmol FdUrd per 10(7) cells. Pretreatment with 50 microM deoxythymidine for 24 hr led to a 2.7-fold increase in the incorporation of this analogue into newly synthesized DNA during the ensuing 16-hr exposure to 0.5 microM [3H]FdUrd. Pretreatment with 0.5 microM methotrexate for 3 hr also increased the [3H]FdUrd incorporation into newly synthesized DNA approximately 5-fold. The coexistence of deoxythymidine or methotrexate with [3H]FdUrd, however, led to decreased incorporation of FdUrd into DNA. More than 50% of the radioactivity in DNA separated by Cs2SO4 equilibrium density gradient centrifugation was proven to be fluorodeoxyuridylate by means of its binding to Lactobacillus casei deoxythymidine monophosphate synthetase.

Fluorodeoxyuridine (FdUrd) is a cytotoxic analogue of thymidine which requires activation by thymidine kinase to FdUMP. FdUMP inhibits thymidylate synthetase and, thus, the synthesis of dTTP. 5'-Aminothymidine (5'-AdThd) can antagonize the feedback inhibition exerted by dTTP on thymidine kinase activity and thereby stimulate FdUrd phosphorylation. This provided a novel approach to assess the degree to which end product inhibition regulates the phosphorylation of FdUrd. We used 5'-AdThd to investigate the effects of dThd and IdUrd on the regulation of FdUrd uptake in intact 647V cells, a human bladder cancer cell line. Contributions from catabolic processes were found not to be important in our system. We detected no nucleoside phosphorylase activity in the 647V cells or any effect of 5'-AdThd on the breakdown of 5-fluorodeoxyuridine monophosphate to FdUrd by crude preparations from these cells. Thus, phosphorylation by thymidine kinase determined FdUrd uptake (phosphorylation). In the absence of added nucleosides the rate of FdUrd uptake increased in a time dependent fashion. Diminished feedback inhibition of thymidine kinase appeared to be an important factor, as evidenced by a decrease in intracellular dTTP pools and a time dependent loss in the ability of 5'-AdThd to stimulate FdUrd uptake. Thymidine and iododeoxyuridine inhibited FdUrd phosphorylation (uptake) by two mechanisms: competition for the active site of thymidine kinase and increased feedback inhibition. Increased feedback inhibition was indicated by stimulation of FdUrd uptake by 5'-AdThd. The effects of IdUrd on FdUrd uptake were also time dependent, presumably reflecting accumulation of iododeoxyuridine triphosphate and dTTP pools. FdUrd cytotoxicity was modulated by dThd, IdUrd, and 5'-AdThd in parallel to their perturbation of FdUrd uptake. Individually they reduced the growth inhibitory properties of FdUrd. These results show that the regulation of FdUrd uptake is critically dependent on the presence of dThd and IdUrd and emphasize the potential importance of circulating levels of these nucleosides in mediating FdUrd activation and cytotoxicity.

In vitro resistance of HCT-8 cells to 5-fluoro-2'-deoxyuridine (FdUrd) has been obtained after a stepwise increase (up to 1 microM) in the concentration of the nucleoside in the culture medium over a period of 6 months. With a clonogenic assay, the toxicities of 17 antineoplastic agents on HCT-8-sensitive and -resistant cells were compared. Resistant cells were 700-fold resistant to FdUrd and showed different degrees of cross-resistance to several purine and pyrimidine nucleoside analogues; no cross-resistance was noted to base analogues and other cytotoxic drugs. The activities of FdUrd phosphorylase, 5'-fluorouridine kinase, 5-fluorouridine phosphorylase, 5-fluorouracil phosphoribosyltransferase, and thymidylate synthase were not significantly different in the sensitive and resistant cell lines. Mixing experiments indirectly excluded the possible elevation of the level of cytoplasmic phosphatases. The activity of FdUrd kinase in sensitive cell extracts was no more than twice that of resistant cells, and the affinities of this enzyme for FdUrd and thymidine at 0.1 to 50 microM were similar in both cell lines. However, cultures of this line failed to accumulate 5-fluoro-2'-deoxyuridylate at concentrations of FdUrd that resulted in substantial accumulation of the nucleotide in the sensitive line. These contrasting data suggested a defect in the facilitated diffusion of the analogue. The entrance of free nucleoside and its subsequent phosphorylation were compared in the two lines over short (2 to 40 s) and longer time periods at 25 degrees C and at 4 degrees C over a range of extracellular FdUrd concentrations (0.1 to 10 microM). Rapid entrance of the nucleoside into sensitive cells was observed, but entry was not detectable in resistant cells. Dipyridamole and nitrobenzylthioinosine inhibition as well as high-performance liquid chromatography analysis confirmed that data obtained from the sensitive cell line during the first 40 s primarily reflected facilitated diffusion of free nucleoside.

The metabolic products formed and incorporated into the nucleic acids (RNA and DNA) of mice bearing Lewis lung carcinoma (LLC) following optimal doses of 5-fluorouracil (FUra), 5-fluoro-2'-deoxyuridine (FdUrd), and 5-fluoro-2'-deoxycytidine (FdCyd) coadministered with tetrahydrouridine (H4Urd), a potent inhibitor of cytidine deaminase, were examined. Treatment with FdCyd plus H4Urd resulted in a tumor-selective incorporation and formation of antimetabolites compared to either FUra or FdUrd treatments. Between 45- and greater than 5400-fold higher levels of the potent thymidylate synthetase inhibitor, 5-fluoro-2'-deoxyuridylate (FdUMP), were formed in tumor than in any of the normal tissues analyzed. RNA-level antimetabolites (FUra, 5-fluorouridine, and 5-fluorouridylate) were also between 3 and greater than 990-fold higher in tumor compared to normal tissue following FdCyd plus H4Urd administration. DNA-level antimetabolites (FdCyd, 5-fluorodeoxycytidylate, FdUrd, and FdUMP) were from 2- to 6-fold higher in tumor compared to normal tissue. FUra and FdUrd treatments resulted in between 3 and greater than 1300-fold higher RNA-level antimetabolites and from 4 to greater than 1020-fold higher FdUMP pools in normal tissues than FdCyd plus H4Urd treatment. DNA-level antimetabolites were also from 4- to 32-fold higher in normal tissues following optimal doses of FUra or FdUrd. In tumor tissue, optimal doses of FUra or FdUrd resulted in lower (a) FdUMP levels (5- to 2-fold), (b) RNA-level antimetabolites (6- to 3-fold), and (c) DNA-level antimetabolites (10- to 4-fold) compared to an optimal dosage of FdCyd plus H4Urd. In serum, the administration of H4Urd resulted in the protection of FdCyd from systemic catabolism, unlike that found with FUra or FdUrd. Substantial levels of FdUMP, FUrd, and FUMP were noted in serum following FUra or FdUrd treatment. The formation of di- and triphosphate antimetabolite pools and the incorporation of antimetabolites into the RNA and DNA of normal and tumor tissues demonstrated trends similar to those mentioned above with nucleoside, mononucleotide, and free base pools. H4Urd treatment of 25 mg/kg did not affect the elevated levels of deoxycytidine kinase or deoxycytidylate deaminase in LLC tumor tissue or the low levels found in normal tissue. A critical feature of this chemotherapeutic strategy using FdCyd plus H4Urd was that the elevated level of cytidine deaminase in LLC tumor tissue was inhibited less than 10% by the administration of 25 mg/kg H4Urd, whereas deoxycytidine deaminase activities in normal tissues (including bone marrow and intestine) were inhibited greater than 93%.(ABSTRACT TRUNCATED AT 400 WORDS)

The following parameters were evaluated at several points throughout unperturbed suspension culture growth of L1210 cells: cell volume; DNA histograms; the mean content of cellular DNA, RNA, and protein; ribonucleoside and deoxyribonucleoside triphosphate pools; phosphoribosyl pyrophosphate; and the incorporation of glycine into purine bases. The cell volume, the incorporation of glycine into purine bases, and the intracellular pools of phosphoribosyl pyrophosphate and dexoyribunucleotides began to decrease significantly during the midportion of logarithmic cell growth. However, there was no significant change in the DNA content per cell during culture growth. The RNA, protein content, and ribonucleotides all demonstrated a biphasic pattern with the highest values obtained during the midportion of logarithmic growth followed by rapid decline as the culture approached plateau growth. These intracellular fluctuations in de novo synthesis and precursor pools were correlated with the variable intracellular accumulation of three fluoropyrimidines (5-fluorouracil, 5-fluorouridine, and 5-fluorodeoxyuridine) and their active metabolites (5-fluorouridine triphosphate and 5-fluorodeoxyuridylate). These studies were performed to demonstrate that multiple biochemical alterations occur during logarithmically growing suspension cell cultures and could result in misleading conclusions of experiments with antimetabolites unless these factors are considered in the context of the performed studies.

We have recently demonstrated that the major biliary metabolite of 5-fluorouracil (FUra) in cancer patients is a conjugate of the FUra catabolite 2-fluoro-beta-alanine (FBAL) and cholic acid (D.J. Sweeny, S. Barnes, G. Heggie, and R.B. Diasio. Proc. Natl. Acad. Sci. USA, 84:5439-5443, 1987). This finding prompted us to further examine the metabolism and biliary excretion of clinically relevant concentrations of the fluoropyrimidines FUra and 5-fluoro-2'-deoxyuridine (FdUrd) using the isolated perfused rat liver. During infusion of fluoropyrimidines, rates of appearance of metabolites in bile were similar with both 1 microM FUra and 1 microM FdUrd but were 9-fold higher with 25 microM FUra. Analysis by high performance liquid chromatography demonstrated that unmetabolized fluoropyrimidines and known catabolites (i.e., FBAL) accounted for less than 15% of the total metabolites in bile and that the majority of the biliary metabolites were eluted as three distinct nonpolar compounds. Fast atom bombardment-mass spectrometry demonstrated that these unique metabolites had molecular weights of 497 (peak 1), 497 (peak 2), and 481 (peak 3). These metabolites were hydrolyzed by cholylglycine hydrolase to FBAL and unconjugated bile acids that were identified by gas chromatography-mass spectrometry to be alpha-muricholic acid (peak 1), cholic acid (peak 2), and chenodeoxycholic acid (peak 3). Thus, the major biliary metabolites of FUra and FdUrd were identified as N-(bile acid)-FBAL conjugates. While the N-(bile acid)-FBAL conjugates were the major metabolites in bile, dihydroFUra was the major (greater than 70%) metabolite eliminated into perfusate. In summary, these results demonstrate that FUra and FdUrd undergo similar metabolism in the isolated perfused rat liver and, as was observed in humans, the major biliary fluoropyrimidine metabolites are conjugates of FBAL and bile acids.

The mouse T-lymphoma (S-49) cell line is useful for individually studying RNA- and DNA-directed effects of 5-fluoropyrimidines. On the basis of their metabolic activation, biochemical effects on pyrimidine nucleotide metabolism, and biological toxicity, we hve established that incubation of S-49 cells with 5-fluorodeoxyuridine produces only DNA-directed toxicity (thymidylate synthetase inhibition), incubation with 5-fluorouracil (FUra) + thymidine only RNA-directed toxicity, and incubation with FUra alone produces both DNA- and RNA-directed toxicity. The DNA component of 5-fluoropyrimidine toxicity causes immediate growth inhibition of asynchronous S-49 cell cultures, which is self-limited within 12 hr both by the accumulation of intracellular deoxyuridine 5'-monophosphate competing for thymidylate synthetase binding and by the excretion of deoxyuridine into the cell medium which competes with 5-fluorodeoxyuridine uptake. The RNA-directed component causes growth inhibition and cell kill after a delay of 1 doubling time in asynchronous cultures. Studies with cells synchronized by centrifugal elutriation indicate that the RNA-directed FUra effects are expressed only in the G1 phase of the cell cycle and cause rapid cell lysis, while the DNA-directed component is specific to the S phase. Experiments using continuous exposure of synchronized cells to FUra alone demonstrate that the activities of the RNA- and DNA-directed components interact with each other. Specifically, DNA-directed toxicity arrests cells in S phase, preventing them from progressing into G1 where RNA-directed toxicity is expressed, which may account for the augmentation of FUra toxicity by thymidine as reported in other systems.

Selective accumulation/retention of 3',5'-dioctanoyl-5-fluoro-2'-deoxyuridine (FdUrd-C8) and sustained release of its active metabolites, 5-fluoro-2'-deoxyuridine (FdUrd) and 5-fluoro-2'-deoxyuridylate (FdUMP), in the rabbit hepatoma (VX-2) were achieved following intrahepatic arterial administration of FdUrd-C8 solution in Lipiodol. Though no significant difference in the FdUrd-C8 levels among the tumor and nontumorous liver was observed immediately after administration, slower elimination of FdUrd-C8 from the tumor (t 1/2 = 15.8 h) than that from nontumorous sites (t 1/2 = 3.8-4.2 h) resulted in selective retention of FdUrd-C8 (17- to 157-fold) in the tumor. Selectively higher levels of FdUrd and FdUMP in the tumor were also achieved (5- to 35-fold) and kept for 72 h after administration. The selective accumulation was also demonstrated in radioactivity distribution after administration of [6-3H]-FdUrd-C8. The ratio of radioactivity in the tumor divided by that in the blood (T/B ratio) was in a range of 870 to 5400 during a 15- to 1440-min period after administration. A trace of radioactivity was found in the stomach, duodenum, kidneys, and bone marrow. Roles of activation and deactivation enzymes on the selective distribution of FdUrd-C8 were also investigated. Esterase activity, which is responsible for the regeneration of FdUrd from FdUrd-C8, was relatively low in the tumor before administration and gradually increased after administration. Phosphorylase activity, which is related to phosphorolytic cleavage of FdUrd, in the tumor was about 3/5 as much as that in the nontumorous liver. These enzyme activities seem to play limited roles in the selective accumulation/retention and regeneration of the drug.

The nucleoside transport inhibitor dipyridamole can increase the cytotoxicity of 5-fluorouracil in a human colon cancer cell line (HCT 116) without affecting the total amount of fluorouracil incorporated into the acid soluble and insoluble fractions (J. L. Grem and P. H. Fischer, Cancer Res., 45: 2967-2972, 1985). We now report that dipyridamole altered the pattern of fluorouracil metabolism and provided a selective increase in intracellular fluorodeoxyuridine monophosphate (FdUMP) levels. At 2 and 4 h after exposure to fluorouracil and dipyridamole, FdUMP levels were approximately 5-fold higher in the presence of dipyridamole. The ratio of FdUMP to fluorouridine triphosphate at 4 h was substantially increased in the presence of dipyridamole (0.4 +/- 0.05) compared to fluorouracil alone (0.08 +/- 0.03). In cells preloaded with fluorodeoxyuridine (FdUrd), dipyridamole potently inhibited the efflux of FdUrd, leading to an increased retention of intracellular FdUMP. One h following removal of [6-3H]FdUrd, the FdUMP levels were increased 8-fold in the presence of dipyridamole, and the half-life of intracellular FdUMP was increased from 24 to 78 min. We have previously shown that the addition of sufficient thymidine (25 microM) can prevent the augmentation of fluorouracil toxicity produced by dipyridamole. In these studies, the addition of 25 microM thymidine reduced the FdUMP levels to less than half of those measured in the presence of fluorouracil plus dipyridamole for the first 8 h of exposure, and reduced the FdUMP levels to 6% of the FdUMP levels seen with fluorouracil and dipyridamole after 24 h of exposure. Thymidine prevented the enhanced intracellular retention of FdUMP produced by dipyridamole in cells preloaded with FdUrd. In addition, thymidine inhibited the accumulation of FdUMP in cells exposed to FdUrd. In cancer cells which significantly catabolize FdUMP, the ability of dipyridamole to block the efflux of FdUrd may provide an effective means of selectively increasing FdUMP levels and enhancing the toxicity of fluorouracil. Furthermore, dipyridamole blocked the efflux of deoxyuridine and prolonged the intracellular half-life of deoxyuridine monophosphate. In cells prelabeled with [2'-3H]dUrd, transfer of tritium to FdUrd and FdUMP occurred in cells exposed to fluorouracil and dipyridamole. These data suggest that blockade of nucleoside efflux can enhance the availability of deoxyribose-1-phosphate donors for the synthesis of FdUrd. Thus, dipyridamole's ability to inhibit nucleoside transport can perturb the metabolism of a nucleobase, fluorouracil.

Six cell lines differing in histological origin were studied regarding the growth inhibitory effect of fluoropyrimidines in relation to their metabolism. The human colon carcinoma cell line WiDr was most sensitive to 5-fluorouracil (FUra) (50% growth inhibitory concentration, 0.7 microM) and to its analogue 5'deoxy-5-fluorouridine (5'dFUR) (50% growth inhibitory concentration, 18 microM). The murine B16 melanoma cell line was moderately sensitive to FUra but least sensitive to 5'dFUR. The 50% growth inhibitory concentration values in the human melanoma cell lines IGR3 and M5, the transformed human intestine cell line intestine 407 and the rat hepatoma cell line H35 varied for FUra between 1.7 and 5.0 microM, and for 5'dFUR between 54 and 160 microM. Several enzymes from pyrimidine metabolism responsible for FUra metabolism were measured with FUra as a substrate. The activity of uridine phosphorylase, which catalyzes the conversion of 5'dFUR to FUra, was lowest in B16 cells correlating with the low sensitivity to 5'dFUR. When adenosine 5'-triphosphate was included in the reaction mixture for uridine phosphorylase, FUra was rapidly channeled into FUra nucleotides via its nucleoside. The rate of channeling appeared to correlate with the nucleoside phosphorylase activity in the various cell lines. In several cell lines activities of nucleotide-degrading enzymes were rather high and interfered with the measurement of orotate phosphoribosyl transferase (OPRT) with FUra as substrate. Addition of the phosphatase inhibitor glycerol-2-phosphate partly prevented breakdown of the newly formed 5-fluorouridine 5'-monophosphate and enabled measurement of OPRT. The WiDr cell line had a relatively high OPRT activity which could explain its sensitivity to FUra. The activity of thymidylate synthase was measured at a suboptimal concentration of 1 microM and at the optimal concentration of 10 microM deoxyuridine 5'-phosphate. With all cell lines the ratio between the activities at 10 and 1 microM was between 2.3 and 3.6. The activity of thymidylate synthase was lowest in WiDr and IGR3 cells and 3-4 times higher in M5 and Intestine 407 cells. The inhibition of 0.01 microM 5-fluorodeoxyuridine 5'-monophosphate was 80-90% at 1 microM deoxyuridine 5'-phosphate and 50-70% at 10 microM deoxyuridine 5'-phosphate with all cell lines. At 0.1 microM 5-fluorodeoxyuridine 5'-monophosphate enzyme activity was inhibited by 95-100%. The incorporation of FUra into RNA was relatively low in IGR3 cells and 3-5 times higher in all other cell lines.(ABSTRACT TRUNCATED AT 400 WORDS)