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Objective: Desmoplasia and hypovascularity are thought to impede drug delivery in pancreatic ductal adenocarcinoma (PDAC). However, stromal depletion approaches have failed to show clinical responses in patients. Here, we aimed to revisit the role of the tumour microenvironment as a physical barrier for gemcitabine delivery., Design: Gemcitabine metabolites were analysed in LSL-Kras G12D/+ ; LSL-Trp53 R172H/+ ; Pdx-1-Cre (KPC) murine tumours and matched liver metastases, primary tumour cell lines, cancer-associated fibroblasts (CAFs) and pancreatic stellate cells (PSCs) by liquid chromatography-mass spectrometry/mass spectrometry. Functional and preclinical experiments, as well as expression analysis of stromal markers and gemcitabine metabolism pathways were performed in murine and human specimen to investigate the preclinical implications and the mechanism of gemcitabine accumulation., Results: Gemcitabine accumulation was significantly enhanced in fibroblast-rich tumours compared with liver metastases and normal liver. In vitro, significantly increased concentrations of activated 2',2'-difluorodeoxycytidine-5'-triphosphate (dFdCTP) and greatly reduced amounts of the inactive gemcitabine metabolite 2',2'-difluorodeoxyuridine were detected in PSCs and CAFs. Mechanistically, key metabolic enzymes involved in gemcitabine inactivation such as hydrolytic cytosolic 5'-nucleotidases (Nt5c1A, Nt5c3) were expressed at low levels in CAFs in vitro and in vivo, and recombinant expression of Nt5c1A resulted in decreased intracellular dFdCTP concentrations in vitro. Moreover, gemcitabine treatment in KPC mice reduced the number of liver metastases by >50%., Conclusions: Our findings suggest that fibroblast drug scavenging may contribute to the clinical failure of gemcitabine in desmoplastic PDAC. Metabolic targeting of CAFs may thus be a promising strategy to enhance the antiproliferative effects of gemcitabine., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Development of gemcitabine (GEM) nanocarriers as theranostic agents for pancreatic cancer chemotherapy has received extensive attention in recent years. A novel enzyme-sensitive albumin-based GEM delivery nanoplatform was developed in this research by simple conjugation of GEM to human serum albumin (HSA) via cathepsin B cleavable peptide GFLG and then complexing with near-infrared (NIR) dye IR780, forming a HSA-GEM/IR780 complex. The successful preparation of HSA-GEM/IR780 complex was confirmed by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS), UV-vis-NIR absorption spectra and fluorescent emission spectra. The in vivo performance of HSA-GEM/IR780 complex was carried out on BxPC-3 pancreatic tumor xenografted mice. As revealed by in vivo NIR imaging, HSA-GEM/IR780 exhibited enhanced accumulation and long-term retention in tumor tissues compared to free IR780. Meanwhile, compared to free GEM, the deamination of GEM nanovectors into inactive 2',2'-difluorodeoxyuridine (dFdU) can be greatly suppressed, while the concentration of the activated form of GEM (gemcitabine triphosphate, dFdCTP) was significantly increased in tumor tissue, thus exhibiting superior tumor inhibition activity with minimal side effects., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Antimetabolites are incorporated into DNA and RNA, affecting their function. Liquid-chromatography-mass-spectrometry (LC-MS-MS) permits the sensitive, selective analysis of normal nucleosides. The method was adapted to measure the incorporation of deoxyuridine, gemcitabine (difluorodeoxycytidine), its metabolite difluorodeoxyuridine (dFdU), and the novel compound fluorocyclopentenylcytosine (RX3117). DNA was degraded to its deoxynucleotides for quantification by LC-MS-MS, gradient chromatography on a Phenomenex prodigy-3-ODS with positive ionization. The range of deoxyuridine DNA-mis-incorporation varied nine-fold in 27 cell lines (leukemia, colon, ovarian, lung cancer). At low-folate conditions a 2.1-fold increase in deoxyuridine was observed. Global methylation (given as % 5-methyl-deoxycytidine) was comparable between the cell lines (4.6-6.5%). Exposure of A2780 cells to 1 μM gemcitabine (4 hours) resulted in 3.6 pmol gemcitabine/μg DNA, but in AG6000 cells (deoxycytidine-kinase-deficient) no incorporation was found. However, when A2780, AG6000, or CCRF-CEM cells were exposed to 100 μM dFdU we found it as gemcitabine, 20.5, 19.6, and 0.51 pmol gemcitabine/μg DNA, respectively. Preincubation of CCRF-CEM cells with cyclopentenyl-cytosine (a CTP-synthetase inhibitor) increased dFdU incorporation four-fold. Apparently dFdU is activated independently of deoxycytidine-kinase and possibly converted in-situ to dFdCMP. RX3117 was incorporated into both DNA and RNA (0.0037 and 0.00515 pmol/μg, respectively). In summary, a sensitive method to quantify the incorporation of gemcitabine, deoxyuridine, and RX-3117 was developed, which revealed that dFdU was incorporated into DNA as the parent compound gemcitabine.

The goal of this study was to understand roles of nucleoside and nucleobase transport processes in capecitabine pharmacology in cells derived from human renal proximal tubule cells (hRPTCs) and three human renal cell carcinoma (RCC) cell lines, A498, A704, and Caki-1. Human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) mediated activities and a sodium-independent nucleobase activity were present in hRPTCs. In hRPTCs, uptake of 5'-deoxy-5-fluorouridine (DFUR), a nucleoside metabolite of capecitabine, was pH dependent with highest uptake seen at pH 6.0. In RCC cell lines, hENT1 was the major nucleoside transporter. Nucleobase transport activity was variable among the three RCC cell lines, with Caki-1 showing the highest and A498 showing the lowest activities. Treatment of RCC cell lines with interferon alpha (IFN-α) increased thymidine phosphorylase levels and prior treatment of RCC cell lines with IFN-α followed by 5-FU or DFUR resulted in enhanced sensitivity of all cell lines to 5-FU and two of three cell lines to DFUR. We report for the first time a nucleobase transport activity in hRPTCs and RCC cell lines. In addition, our in vitro cytotoxicity results showed that RCC cell lines differed in their response to 5-FU and DFUR and prior treatment with IFN-α potentiated cytotoxic response to metabolites of capecitabine.

In this study we report a high sensitive method for the simultaneous analysis of LY2334737 (2'-deoxy-2',2'-difluoro-N-(1-oxo-2-propylpentyl)-cytidine), an amide prodrug of gemcitabine (2', 2'-difluoro-deoxycytidine), along with its active drug gemcitabine and its major metabolite dFdU (2',2'-difluoro-deoxyuridine) by LC-MS/MS. Quantification of all three analytes within a single analysis was challenging because the physio-chemical properties of LY2334737 were significantly different from gemcitabine and dFdU and was accomplished by incorporating column-switching. The assay was fully validated to quantify LY2334737 from 0.1 to 100ng/mL, gemcitabine from 0.25 to 100ng/mL and dFdU from 1 to 1000ng/mL in order to cover the diverse concentration ranges expected in clinical samples. A 25-fold dilution was also validated to accommodate any samples outside this range. Overall, the assay had good accuracy (ranging from -7.0 to 1.2% relative error) and precision (ranging from 2.1 to 8.4% relative standard deviation). Extraction efficiency was greater than 80% for all three analytes and there were no matrix effects. Plasma samples were stable for 24h at room temperature, 660 days in frozen storage, and at least 4 freeze-thaw cycles, at both -20 and -70°C. Data from clinical trials showed that plasma concentrations for LY2334737, gemcitabine, and dFdU were successfully quantified from a single LC-MS/MS analysis and that the assay ranges selected for the three analytes were appropriate and minimized the need for reanalysis., (Copyright © 2013. Published by Elsevier B.V.)

Purpose: Multiple proteins are involved in activation and inactivation of 2',2'-difluorodeoxycytidine (gemcitabine, dFdC). We aimed to clarify the mechanism of dFdC resistance in a pancreatic cancer cell line by applying a combination of targeted proteomic and metabolomic analyses., Methods: Twenty-five enzyme and transporter proteins and 6 metabolites were quantified in sensitive and resistant pancreatic cancer cell lines, PK9 and RPK9, respectively., Results: The protein concentration of deoxycytidine kinase (dCK) in RPK9 cells was less than 0.02-fold (2 %) compared with that in PK9 cells, whereas the differences (fold) were within a factor of 3 for other proteins. Targeted metabolomic analysis revealed that phosphorylated forms of dFdC were reduced to less than 0.2 % in RPK9 cells. The extracellular concentration of 2',2'-difluorodeoxyuridine (dFdU), an inactive metabolite of dFdC, reached the same level as the initial dFdC concentration in RPK9 cells. However, tetrahydrouridine treatment did not increase phosphorylated forms of dFdC and did not reverse dFdC resistance in RPK9 cells, though this treatment inhibits production of dFdU., Conclusions: Combining targeted proteomics and metabolomics suggests that acquisition of resistance in RPK9 cells is due to attenuation of dFdC phosphorylation via suppression of dCK.

Aim: The aim of this study was to evaluate the association of gemcitabine pathway SNPs with detailed pharmacokinetic measures obtained from solid tumor patients receiving gemcitabine-based therapy., Materials & Methods: SNPs within nine gemcitabine pathway genes, namely CDA, CMPK, DCK, DCTD, NT5C2, NT5C3, SLC28A1, SLC28A3 and SLC29A1 were analyzed for association with gemcitabine pharmacokinetics., Results: Significant association of gemcitabine clearance with SNPs in NT5C2 was identified. Clearance of 2´,2´-difluorodeoxyuridine, a gemcitabine metabolite was significantly predicted by CDA, SLC29A1 and NT5C2 SNPs. This study reports an association of formation clearance of 2´,2´-difluoro-2´-deoxycytidine triphosphate, an active form of gemcitabine with SNPs within uptake transporters SLC28A1, SLC28A3 and SLC29A1., Conclusion: Genetic variation in gemcitabine pathway genes is associated with its pharmacokinetics and hence could influence gemcitabine response. Our study identified pharmacogenetic markers that could be further tested in larger patient cohorts and could open up opportunities to individualize therapy in solid tumor patients.

Gemcitabine is an established chemotherapy agent in several solid tumors. Its mechanism of action has been theoretically established and this is supported with strong experimental evidence. However, certain aspects of the resistance mechanism for this agent remain elusive. We present a method of analysis using tandem liquid chromatography and mass spectrometry that provides a broader, yet more focused view of the action of gemcitabine and its primary metabolite, difluorodeoxyuridine in relation to the (deoxy) nucleoside and (deoxy) nucleotide pools in tumor cell lines. Alcoholic cytosole extracts were incubated with alkaline phosphatase reducing the nucleotide pools to their respective nucleosides. Determination of the nucleoside content by a sensitive LCMSMS method before and after incubation enables the calculation of the total amount of phosphorylation of each (deoxy) nucleoside in the cell. Incubation with clinically relevant levels of gemcitabine (dFdC) or difluorodeoxyuridine (dFdU) for 24 hours enabled the determination of the changes in the (deoxy) nucleotide pools in relation to chemotherapeutic and toxicological effects. Confirmation of the presence of dFdC phosphorylation is presented as well as direct evidence of dFdU phosphorylation after both dFdC and dFdU treatment. Differences in the nucleotide pools are presented after dFdC and dFdU incubation, indicating that dFdU might have more chemotherapeutic properties than previously believed.

Purpose: To develop a sensitive analytical method to quantify gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and its metabolites 2',2'-difluorodeoxyuridine (dFdU) and 2',2'-difluorodeoxycytidine-5'-triphosphate (dFdCTP) simultaneously from tumour tissue., Methods: Pancreatic ductal adenocarcinoma tumour tissue from genetically engineered mouse models of pancreatic cancer (KP ( FL/FL ) C and KP ( R172H/+) C) was collected after dosing the mice with gemcitabine. (19)F NMR spectroscopy and LC-MS/MS protocols were optimised to detect gemcitabine and its metabolites in homogenates of the tumour tissue., Results: A (19)F NMR protocol was developed, which was capable of distinguishing the three analytes in tumour homogenates. However, it required at least 100 mg of the tissue in question and a long acquisition time per sample, making it impractical for use in large PK/PD studies or clinical trials. The LC-MS/MS protocol was developed using porous graphitic carbon to separate the analytes, enabling simultaneous detection of all three analytes from as little as 10 mg of tissue, with a sensitivity for dFdCTP of 0.2 ng/mg tissue. Multiple pieces of tissue from single tumours were analysed, showing little intra-tumour variation in the concentrations of dFdC or dFdU (both intra- and extra-cellular). Intra-tumoural variation was observed in the concentration of dFdCTP, an intra-cellular metabolite, which may reflect regions of different cellularity within a tumour., Conclusion: We have developed a sensitive LC-MS/MS method capable of quantifying gemcitabine, dFdU and dFdCTP in pancreatic tumour tissue. The requirement for only 10 mg of tissue enables this protocol to be used to analyse multiple areas from a single tumour and to spare tissue for additional pharmacodynamic assays.

Gemcitabine (dFdC) is a chemotherapeutic nucleoside analog that undergoes uptake via equilibrative nucleoside transporters (hENT) followed by sequential phosphorylation to the active triphosphate moiety (dFdCTP). Its deaminated metabolite, 2',2'-difluorodeoxyuridine (dFdU), competes with the parent compound for cellular entry via hENTs, but over time dFdU increases the net intracellular accumulation of dFdC by a currently unknown mechanism. In this study, we investigated whether dFdU affects intracellular phosphorylation of gemcitabine by modulating the activity of deoxycytidine kinase (dCK). We report here that coincubation of dFdU with dFdC significantly increases intracellular levels of dFdCTP. dFdCTP was not identified as a substrate for hENTs, suggesting that dFdU affects the formation rather than elimination of the triphosphate. To further characterize the disposition of dFdC in the presence of dFdU, the net intracellular radioactivity of [5-(3)H]dFdC and corresponding metabolic profile were evaluated in HeLa cells transfected with dCK-targeting small interfering RNA. Intracellular radioactivity significantly decreased in cells with compromised intracellular phosphorylation, which was mainly due to a loss in dFdCTP. Although dFdU increased the net intracellular radioactivity of [5-(3)H]dFdC at 24 h in control cells, this increase was abolished in the absence of dCK activity, strongly suggesting that the interaction between dFdU and dFdC occurs via modulation of both transport and metabolism. In conclusion, we have demonstrated that the intracellular distribution of dFdC is dependent on both transport and metabolic processes, and that by affecting the rate at which dFdC enters the cell, the presence of dFdU may be altering the metabolic fate of the parent compound (dFdC).

Purpose: We investigated single-nucleotide polymorphisms of the cytidine deaminase gene (CDA), which encodes an enzyme that metabolizes gemcitabine, to clarify the relationship between the single-nucleotide polymorphism 208G>A and the pharmacokinetics and toxicity of gemcitabine in cancer patients treated with gemcitabine plus cisplatin., Experimental Design: Six Japanese cancer patients treated with gemcitabine plus cisplatin were examined. Plasma gemcitabine and its metabolite 2',2'-difluorodeoxyuridine were measured using an high-performance liquid chromatography method, and the CDA genotypes were determined with DNA sequencing., Results: One patient, a 45-year-old man with pancreatic carcinoma, showed severe hematologic and nonhematologic toxicities during the first course of chemotherapy with gemcitabine and cisplatin. The area under the concentration-time curve value of gemcitabine in this patient (54.54 microg hour/mL) was five times higher than the average value for five other patients (10.88 microg hour/mL) treated with gemcitabine plus cisplatin. The area under the concentration-time curve of 2',2'-difluorodeoxyuridine in this patient (41.58 microg hour/mL) was less than the half of the average value of the five patients (106.13 microg hour/mL). This patient was found to be homozygous for 208A (Thr70) in the CDA gene, whereas the other patients were homozygous for 208G (Ala70)., Conclusion: Homozygous 208G>A alteration in CDA might have caused the severe drug toxicity experienced by a Japanese cancer patient treated with gemcitabine plus cisplatin.

Capecitabine, an anticancer prodrug, is thought to be biotransformed into active 5-fluorouracil (5-FU) by three enzymes. After oral administration, capecitabine is first metabolized to 5'-deoxy-5-fluorocytidine (5'-DFCR) by carboxylesterase (CES), then 5'-DFCR is converted to 5'-deoxy-5-fluorouridine (5'-DFUR) by cytidine deaminase. 5'-DFUR is activated to 5-FU by thymidine phosphorylase. Although high activities of drug metabolizing enzymes are expressed in human liver, the involvement of the liver in capecitabine metabolism is not fully understood. In this study, the metabolism of capecitabine in human liver was investigated in vitro. 5'-DFCR, 5'-DFUR, and 5-FU formation from capecitabine were investigated in human liver S9, microsomes, and cytosol in the presence of the inhibitor of dihydropyrimidine dehydrogenase, 5-chloro-2,4-dihydroxypyridine. 5'-DFCR, 5'-DFUR, and 5-FU were formed from capecitabine in cytosol and in the combination of microsomes and cytosol. Only 5'-DFCR formation was detected in microsomes. The apparent K(m) and V(max) values of 5-FU formation catalyzed by cytosol alone and in combination with microsomes were 8.1 mM and 106.5 pmol/min/mg protein, and 4.0 mM and 64.0 pmol/min/mg protein, respectively. The interindividual variability in 5'-DFCR formation in microsomes and cytosol among 14 human liver samples was 8.3- and 12.3-fold, respectively. Capecitabine seems to be metabolized to 5-FU in human liver. 5'-DFCR formation was exhibited in cytosol with large interindividual variability, although CES is located in microsomes in human liver. In the present study, it has been clarified that the cytosolic enzyme would be important in 5'-DFCR formation, as is CES.

A sensitive, accurate and reproducible procedure has been developed for the quantitative determination of gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and its metabolite 2',2'-difluorodeoxyuridine (2dFdU) in human urine. The samples (2 mL) were extracted by solid-phase extraction (SPE) and analyzed by reversed-phase high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC/MS/MS), operating in multiple reaction monitoring (MRM mode). This procedure was validated using 2'-deoxycytidine as internal standard (IS). The urine assay was linear over the range 0-50 microg/L, with a limit of quantification (LLOQ) of 0.2 microg/L for gemcitabine and 1.0 microg/L for the metabolite. The respective limits of detection (LODs) for dFdC and 2dFdU were 0.05 and 0.3 microg/L. The precision and accuracy of the assay were determined on three different days. The within-series precision was found to be always less than 8.5 and 12.7% for gemcitabine and 2dFdU, respectively. The overall precision expressed as relative standard deviation (CVr) was always less than 7.1% for both analytes. The recovery of gemcitabine was always greater than 90% with a CVr <6.3%. The measurement uncertainty determined from the validation data assessed the possibility of determining this drug and its metabolite at trace levels in urine, considering that the combined uncertainty of the whole procedure was always less than 30%., (Copyright 2004 John Wiley & Sons, Ltd.)

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.

Apatmers are synthesized using 2'-fluoropyrimdines in place of normal pyrmidines to stabilize them against enzymatic degradation, and thereby improve their therapeutic efficacy. Despite this stabilizing effect, the apatmers can still be degraded by nucleases in the blood. Primer template extension studies have demonstrated that mammalian DNA polymerases can incorporate these 2'-fluoropyrimidines into growing strands of DNA. The toxicologic effects of these compounds have been examined in rats and woodchucks, animals known to be susceptible to the toxic effects of other modified pyrimidines. Whether these nucleosides can be incorporated into DNA in vivo has not been established. These studies report the development of methodologies and the results of studies designed to determine if and to what extent 2'-fluoropyrimidines are incorporated into tissue DNA following long-term treatment. Rats were dosed intravenously with either 2'-fluorouridine (2'-FU) or 2'-fluorocytidine (2'-FC) at doses of 5, 50, and 500 mg/kg/day for 90 days. Woodchucks were dosed intravenously with either 2'-FU or 2'-FC at doses of 0.75 or 7.5 mg/kg/day for 90 days. The amounts of 2'-FU or 2'-FC in DNA and RNA were quantified using newly developed LC/MS/MS methodologies. Administration of 2'-FU to rats and woodchucks resulted in incorporation of the compound into DNA from liver, spleen, testis, muscle, and kidney. Incorporation also occurred in RNA from rat liver (only tissue examined). Similarly, administration of 2'-FC to rats and woodchucks resulted in incorporation into liver DNA (only tissue examined). These data demonstrate that 2'-fluoropyrimidines are incorporated into DNA and RNA of various tissues of rats and woodchucks following long-term administration.

Capecitabine (N4-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine) is a novel oral fluoropyrimidine carbamate, which is converted to 5-fluorouracil (5-FU) selectively in tumours through a cascade of three enzymes. The present study investigated tissue localisation of the three enzymes in humans, which was helpful for us to design the compound. Carboxylesterase was almost exclusively located in the liver and hepatoma, but not in other tumours and normal tissue adjacent to the tumours. Cytidine (Cyd) deaminase was located in high concentrations in the liver and various types of solid tumours. Finally, thymidine phosphorylase (dThdPase) was also more concentrated in various types of tumour tissues than in normal tissues. These unique tissue localisation patterns enabled us to design capecitabine. Oral capecitabine would pass intact through the intestinal tract, but would be converted first by carboxylesterase to 5'-deoxy-5-fluorocytidine (5'-dFCyd) in the liver, then by Cyd deaminase to 5'-deoxy-5-fluorouridine (5'-dFUrd) in the liver and tumour tissues and finally by dThdPase to 5-FU in tumours. In cultures of human cancer cell lines, the highest level of cytotoxicity was shown by 5-FU itself, followed by 5'-dFUrd. Capecitabine and 5'-dFCyd had weak cytotoxic activity only at high concentrations. The cytotoxicity of the intermediate metabolites 5'-dFCyd and 5'-dFCyd was suppressed by inhibitors of Cyd deaminase and dThdPase, respectively, indicating that these metabolites become effective only after their conversion to 5-FU. Capecitabine, which is finally converted to 5-FU by dThdPase in tumours, should be much safer and more effective than 5-FU, and this was indeed the case in the HCT116 human colon cancer and the MX-1 breast cancer xenograft models.

Protected 2'-deoxy-2'-fluorouridine and 2'-deoxy-2'-fluorocytidine suitable for incorporation into oligonucleotides via the phosphoramidite approach have been prepared. Five modified and two unmodified oligonucleotides have been synthesized to investigate the regiospecific cleavage of a 5S RNA from Escherichia coli by RNase H. In order to show whether the modified oligonucleotides are able to hybridize with the RNA the physico-chemical properties (melting curves, CD spectra) of analogous DNA/oligodeoxyribonucleotide duplexes have been examined. The modified oligonucleotides are shown to form stable duplexes with a DNA-matrix which exist in an A-like form. Two of the modified probes containing four 2'-deoxy-2'-fluorocytidines or two 2'-deoxy-2'-fluorouridines direct the splitting by RNase H of only one phosphodiester bond of the RNA.

N4-Trimethoxybenzoyl-5'-deoxy-5-fluorocytidine (Ro 09-1390), a prodrug of the cytostatic 5'-deoxy-5-fluorouridine (5'-DFUR), was synthesized with the aim of reducing of the dose-limiting toxicity of 5'-DFUR, which is diarrhea. In mice bearing Lewis lung carcinoma, 5'-DFUR given po produced a substantial amount of 5-fluorouracil (5-FU) in the intestinal tract as well as tumors, where the enzyme pyrimidine nucleoside phosphorylase, essential for conversion of 5'-DFUR to 5-FU, is predominantly located. With the oral administration of Ro 09-1390 only a small amount of 5-FU was formed in the intestine; however, the administration of Ro 09-1390 and 5'-DFUR at the same dose produced similar amounts of 5-FU in tumor tissues. These differences in metabolism were reflected in their toxicity and antitumor efficacy. The administration of 5'-DFUR resulted in damage to the intestinal mucosal membrane and diarrhea in normal mice, whereas Ro 09-1390 was much less toxic to the intestinal tract. As regards antitumor activity, Ro 09-1390 and 5'-DFUR at equivalent doses inhibited the growth of Lewis lung carcinoma to similar extents. Since Ro 09-1390 was much less toxic to the intestinal tract than 5'-DFUR, mice bearing Lewis lung carcinoma could be given Ro 09-1390 daily over a longer period and at a higher dose, resulting in a longer survival time.

The modes of action of 5-fluoro-2'-deoxyuridine (FdUrd) and 5-fluoro-2'-deoxycytidine (FdCyd) were studied in PHA-stimulated lymphocytes from normal volunteer donors and a fragile X patient. In both cell types, FdUrd and FdCyd inhibited cell proliferation at concentrations of 3 x 10(-8) M. Thymidylate synthetase was identified as the decisive target for the action of both FdUrd and FdCyd, as judged from the following observations: First, addition of thymidine to the culture medium was able to counteract both FdUrd and FdCyd toxicities, whereas addition of dCyd had no observable effect. Second, inhibition of the in situ thymidylate synthetase activity measured as an increase in the level of [3H]-dThd incorporation coincided with the inhibition of cell proliferation. Third, the inhibition of the thymidylate synthetase-dependent incorporation of [3H]-dUrd into newly synthesized DNA coincided with the inhibition of cell proliferation. The effects of FdUrd and FdCyd on the in vitro expression of fragile site Xq27 of fragile X chromosomes was shown to be based on the depletion of the intracellular pool of thymidine-5'-monophosphate (dTMP), as judged from the following observations: First, both the FdUrd- and FdCyd-dependent induction of site Xq27 coincided with the antiproliferative effects of the respective fluoropyrimidines. Second, addition of thymidine (dThd) to the culture medium both prevented the expression of site Xq27 and neutralized the cytotoxicity of FdUrd and of FdCyd. On the basis of these findings, we provide further evidence for the concept that the fragile X site is located in an AT-rich region.

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)