LC-MS/MS method for quantitation of gemcitabine and its metabolite 2',2'-difluoro-2'-deoxyuridine in mouse plasma and brain tissue: Application to a preclinical pharmacokinetic study.

A simple, sensitive, and relatively fast assay was developed and validated for the quantitation of gemcitabine (dFdC) and its major metabolite 2',2'-difluoro-2'-deoxyuridine (dFdU) in mouse plasma and brain tissue. The assay used a small sample (25 µL plasma and 5 mg brain) for extraction by protein precipitation. After dilution of the supernatant extract, 1 µL was injected into HPLC system for reverse phase chromatographic separation with a total run time of 8 min. Chromatographic resolution of dFdC and dFdU was achieved on a Gemini C18 column (50 × 4.6 mm, 3 µm) utilizing gradient elution. Multiple reaction monitoring (MRM) with positive/negative ion switching was performed for detection of dFdC and its internal standard (dFdC-IS) in positive ion mode and dFdU and its IS (dFdU-IS) in negative ion mode. Two calibration curves ranging from 5-2000 ng/mL and 250-50,000 ng/mL were generated for dFdC and dFdU in mouse plasma, respectively. For measurement of dFdC and dFdU in mouse brain tissue, another two curves were used ranging from 0.02 to 40 ng/mg and 1-40 ng/mg, respectively. This assay demonstrated excellent precision and accuracy within day and between days for simultaneous measurement of dFdC and dFdU at all the concentration levels in both matrices. The other parameters such as selectivity, sensitivity, matrix effects, recovery, and storage stability were also assessed for both analytes in each matrix. Compared to the previously reported methods, the sample extraction in the current assay was simplified significantly, and the analysis time was greatly shortened. We successfully applied the validated method to the analysis of dFdC and dFdU in mouse plasma, brain, and brain tumor tissue in a preclinical pharmacokinetic study.

Floxuridine Oligomers Activated under Hypoxic Environment.

Floxuridine oligomers are anticancer oligonucleotide drugs composed of a number of floxuridine residues. They show enhanced cytotoxicity per floxuridine monomer because the nuclease degradation of floxuridine oligomers directly releases highly active floxuridine monophosphate in cells. However, their clinical use is limited by the low selectivity against cancer cells. To address this limitation, we herein report floxuridine oligomer prodrugs that are active under hypoxia conditions, which is one of the distinguishing features of the microenvironment of all solid tumors. We designed and synthesized two types of floxuridine oligomer prodrugs that possess hypoxia-responsive moieties on nucleobases. The floxuridine oligomer prodrugs showed lower cytotoxicity under normoxia conditions (O2 = 20%), while the parent floxuridine oligomer showed similar anticancer effects under hypoxia conditions (O2 = 1%). The floxuridine oligomer prodrug enabled tumor growth suppression in live mice. This would be the first example demonstrating the conditional control of the medicinal efficacy of oligomerized nucleoside anticancer drugs.

Comparative pharmacokinetic study of PEGylated gemcitabine and gemcitabine in rats by LC-MS/MS coupled with pre-column derivatization and MSALL technique.

Gemcitabine is a small molecular antitumor compound used to treat many types of solid tumors. The clinical application of gemcitabine is limited by its short biological half-life, rapid metabolism and poor tumor tissue targeting. The covalent attachment of polyethylene glycol to gemcitabine is a promising technique to overcome these limitations. After PEGylation, PEGylated gemcitabine could be metabolized into gemcitabine and its metabolites in vivo. Due to the scale effect of PEGylated gemcitabine, the DMPK process of the original drug is greatly changed. Therefore, understanding the pharmacokinetic behavior of PEGylated gemcitabine, gemcitabine and the metabolite dFdU in vivo is really important to clarify the antitumoral activity of these compounds. It would also guide the development of other PEGylated drugs. Due to the complex structure and diverse physiochemical property of PEG, direct quantification analysis of PEGylated gemcitabine presented many challenges in terms of assay sensitivity, selectivity, and robustness. In this article, a data-independent acquisition method, MSALL-based approach using electrospray ionization (ESI) coupled quadrupole time of flight mass spectrometry (MS), was utilized for the determination of PEGylated gemcitabine in rat plasma. The technique consists of a Q1 mass window through all the precursor ions, fragmenting and recording all product ions. PEGylated gemcitabine underwent dissociation in collision cell to generate a series of PEG related ions at m/z 89.0604, 133.0868, 177.1129 of 2, 3, 4 repeating ethylene oxide subunits and PEGylated gemcitabine related ions at m/z 112.0514. PEGylated gemcitabine was detected by the high resolution extracted ions based on the specific compound. For gemcitabine and dFdU, the study used derivatization of these high polarity compounds with dansyl chloride to improve their chromatographic retention. This paper describes comparative pharmacokinetic study of PEGylated gemcitabine and gemcitabine in rats by LC-MS/MS coupled with pre-column derivatization and MSALL technique. The results show that PEGylation could reduce the drug clearance of the conjugated compounds and increase the drug plasma half-life. After administration of PEGylated gemcitabine, the exposure of the free gemcitabine in vivo is lower than administration of gemcitabine, which means that PEGylated gemcitabine possesses lower toxicity compared with gemcitabine.

Nucleoside dimers analogues with a 1,2,3-triazole linkage: conjugation of floxuridine and thymidine provides novel tools for cancer treatment. Part II.

The fluorinated nucleoside dimers with a 1,2,3-triazole linkage are novel compounds within the field of bioorganic chemistry. We report on the synthesis and properties of two groups of nucleoside dimers analogs possessing a different arrangement of the 1,4-disubstituted 1,2,3-triazole linkage. Based on analysis of the 3JHH, 3JH1'C2, and 3JH1'C6 we estimated conformational preferences of sugar part and orientation around glycosidic bond. These compounds show moderate anticancer activity, with cytostatic studies in three different cancer cell lines.

Gemcitabine and metabolite pharmacokinetics in advanced NSCLC patients after bronchial artery infusion and intravenous infusion.

PURPOSE: We investigated the safety, pharmacokinetics, and efficacy of gemcitabine administered via bronchial artery infusion (BAI) and IV infusion in advanced NSCLC patients. METHODS: Patients were eligible if they had received at least two prior cytotoxic chemotherapy regimens. Gemcitabine was administered via BAI as 600 mg/m2 on day one of cycle one, followed by IV as 1000 mg/m2 on day eight of cycle one, and IV on days one and eight of all subsequent cycles. Pharmacokinetics for gemcitabine and dFdU metabolite in plasma, and dFdCTP active metabolite in peripheral blood mononuclear cells (PBMC) were evaluated. Intensive pharmacokinetic sampling was performed after BAI and IV infusions during cycle one. RESULTS: Three male patients (age range 59-68 years) were evaluated. All patients responded with stable disease or better. One PR was observed after cycle three, and the remaining had SD. Cmax (mean ± SD) following BAI for gemcitabine, dFdCTP, and dFdU were 7.71 ± 0.13, 66.5 ± 40.6, and 38 ± 6.27 µM and following IV infusion, 17 ± 2.36, 50.8 ± 3.61, and 83.2 ± 12.3 µM, respectively. The AUCinf (mean ± SD) following BAI for gemcitabine, dFdCTP, and dFdU were 6.89 ± 1.2, 791.1 ± 551.2, and 829.9 ± 217.8 µM h and following IV infusion, 12.5 ± 3.13, 584 ± 86.6, and 1394.64 ± 682.2 µM h, respectively. The AUC and Cmax of dFdCTP after BAI were higher than IV. The median OS was 6.27 months. No grade 3 or 4 toxicity was observed. The most common side effects were all grade ≤ 2 involving nausea, vomiting, rigor, thrombocytopenia, and anemia. CONCLUSIONS: Systemic exposure to dFdCTP was higher after BAI than IV in two out of three patients.

Floxuridine Homomeric Oligonucleotides "Hitchhike" with Albumin In†Situ for Cancer Chemotherapy.

Automated attachment of chemotherapeutic drugs to oligonucleotides through phosphoramidite chemistry and DNA synthesis has emerged as a powerful technology in constructing structure-defined and payload-tunable oligonucleotide-drug conjugates. In practice, however, in vivo delivery of these oligonucleotides remains a challenge. Inspired by the systemic transport of hydrophobic payloads by serum albumin in nature, we report the development of a lipid-conjugated floxuridine homomeric oligonucleotide (LFU20) that "hitchhikes" with endogenous serum albumin for cancer chemotherapy. Upon intravenous injection, LFU20 immediately inserts into the hydrophobic cave of albumin to form an LFU20/albumin complex, which accumulates in the tumor by the enhanced permeability and retention (EPR) effect and internalizes into the lysosomes of cancer cells. After degradation, cytotoxic floxuridine monophosphate is released to inhibit cell proliferation.

Simultaneous determination of gemcitabine prodrug, gemcitabine and its major metabolite 2', 2'-difluorodeoxyuridine in rat plasma by UFLC-MS/MS.

To improve bioavailability and provide resistance to deamination, an array of gemcitabine (dFdC) prodrugs carrying the acyl modifications has been successful in the optimization of pharmacokinetic properties of dFdC, but the reports about 4-N-carbobenzoxy-dFdC (Cbz-dFdC), a dFdC prodrug bearing alkyloxycarbonyl modification, are relatively rare. Notably, in vivo enzymatic hydrolysis was an absolutely essential factor for the activation of these prodrugs, which is correlated with the anti-tumor activity. Therefore, detailed metabolism studies of Cbz-dFdC should be carried out for a more authentic pharmacodynamic evaluation. In order to detect the pharmacokinetic characteristics of Cbz-dFdC, a selective, sensitive and accurate method for the simultaneous determination of Cbz-dFdC, along with dFdC and its major metabolite dFdU in rat plasma was developed and validated using UFLC-MS/MS techniques. Column was at 40 °C for separation using an eluent with acetonitrile and 0.1% formic acid, 1 mM ammonium formate at a flow rate of 0.2 mL/min. Detection was performed using ESI source in positive ion selected reaction monitoring mode by monitoring the following ion transitions m/z 398.1 → 202.2 (Cbz-dFdC), m/z 264.1 → 112.0 (dFdC), m/z 265.3 → 113.2 (dFdU) and m/z 246.1 → 112.0 (IS). Analytes were extracted by simple precipitation with acetonitrile containing internal standards followed by liquid-liquid extraction with ethyl acetate. The calibration curves of Cbz-dFdC, dFdC and dFdU were linear in the concentration range of 2 to 500 ng/mL, 2 to 500 ng/mL and 40 to 10,000 ng/mL, respectively. The assay ranges selected for the three analytes were appropriate and minimized the need for reanalysis. All the validation data, such as intra- and inter-day precision, accuracy, selectivity and stability, were within the required limits. In conclusion, the sensitive analytical assay was selective and accurate for the determination of rat plasma concentrations of Cbz-dFdC, dFdC and dFdU from a single LC-MS/MS analysis and well-suited to support pharmacokinetic studies.

Intracellular pharmacokinetics of gemcitabine, its deaminated metabolite 2',2'-difluorodeoxyuridine and their nucleotides.

AIMS: Gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdC) is a prodrug that has to be phosphorylated within the tumour cell to become active. Intracellularly formed gemcitabine diphosphate (dFdCDP) and triphosphate (dFdCTP) are considered responsible for the antineoplastic effects of gemcitabine. However, a major part of gemcitabine is converted into 2',2'-difluoro-2'-deoxyuridine (dFdU) by deamination. In the cell, dFdU can also be phosphorylated to its monophosphate (dFdUMP), diphosphate (dFdUDP) and triphosphate (dFdUTP). In vitro data suggest that these dFdU nucleotides might also contribute to the antitumour effects, although little is known about their intracellular pharmacokinetics (PK). Therefore, the objective of the present study was to gain insight into the intracellular PK of all dFdC and dFdU nucleotides formed during gemcitabine treatment. METHODS: Peripheral blood mononuclear cell (PBMC) samples were collected from 38 patients receiving gemcitabine, at multiple time points after infusion. Gemcitabine, dFdU and their nucleotides were quantified in PBMCs. In addition, gemcitabine and dFdU plasma concentrations were monitored. The individual PK parameters in plasma and in PBMCs were determined. RESULTS: Both in plasma and in PBMCs, dFdU was present in higher concentrations than gemcitabine [mean intracellular area under the concentration-time curve from time zero to 24 h (AUC0-24 h ) 1650 vs. 95 µM*h]. However, the dFdUMP, dFdUDP and dFdUTP concentrations in PBMCs were much lower than the dFdCDP and dFdCTP concentrations. The mean AUC0-24 h for dFdUTP was 312 µM*h vs. 2640 µM*h for dFdCTP. CONCLUSIONS: The study provides the first complete picture of all nucleotides that are formed intracellularly during gemcitabine treatment. Low intracellular dFdU nucleotide concentrations were found, which calls into question the relevance of these nucleotides for the cytotoxic effects of gemcitabine.

Ultra-sensitive LC-MS/MS method for the quantification of gemcitabine and its metabolite 2',2'-difluorodeoxyuridine in human plasma for a microdose clinical trial.

In microdose clinical trials a maximum of 100 µg of drug substance is administered to participants, in order to determine the pharmacokinetic properties of the agents. Measuring low plasma concentrations after administration of a microdose is challenging and requires the use of ulta-sensitive equipment. Novel liquid chromatography-mass spectrometry (LC-MS/MS) platforms can be used for quantification of low drug plasma levels. Here we describe the development and validation of an LC-MS/MS method for quantification of gemcitabine and its metabolite 2',2'-difluorodeoxyuridine (dFdU) in the low picogram per milliliter range to support a microdose trial. The validated assay ranges from 2.5-500 pg/mL for gemcitabine and 250-50,000 pg/mL for dFdU were linear, with a correlation coefficient (r2) of 0.996 or better. Sample preparation with solid phase extraction provided a good and reproducible recovery. All results were within the acceptance criteria of the latest US FDA guidance and EMA guidelines. In addition, the method was successfully applied to measure plasma concentrations of gemcitabine in a patient after administration of a microdose of gemcitabine.

Fibroblast drug scavenging increases intratumoural gemcitabine accumulation in murine pancreas cancer.

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-KrasG12D/+ ; LSL-Trp53R172H/+ ; 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.

Enzyme-sensitive gemcitabine conjugated albumin nanoparticles as a versatile theranostic nanoplatform for pancreatic cancer treatment.

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.

3'-O- and 5'-O-Propargyl Derivatives of 5-Fluoro-2'-Deoxyuridine: Synthesis, Cytotoxic Evaluation and Conformational Analysis.

A series of new 3'-O- and 5'-O-propargyl derivatives of 5-fluoro-2'-deoxyuridine (1-4) was synthesized by means of propargyl reaction of properly blocked nucleosides (2,4), followed by the deprotection reaction with ammonium fluoride. The synthesized propargylated 5-fluoro-2'-deoxyuridine analogues (1-4) were evaluated for their cytotoxic activity in three human cancer cell lines: cervical (HeLa), oral (KB) and breast (MCF-7), using the sulforhodamine B (SRB) assay. The highest activity and the best SI coefficient in all of the investigated cancer cells were displayed by 3'-O-propargyl-5-fluoro-2'-deoxyuridine (1), and its activity was higher than that of the parent nucleoside. The other new compounds exhibited moderate activity in all of the used cell lines.

Preanalytical Stability of Gemcitabine and its Metabolite 2', 2'-Difluoro-2'-Deoxyuridine in Whole Blood-Assessed by Liquid Chromatography Tandem Mass Spectrometry.

Gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) and metabolite (2',2'-difluoro-2'-deoxyuridine, dFdU) quantification is warranted for individualized treatment strategies. Analyte stability is crucial for the validity of such quantification. We therefore studied the impact of the time interval from blood sampling to separation of plasma on gemcitabine stability. Blood from gemcitabine-treated patients was drawn into tetrahydrouridine (THU)-spiked heparin and ethylenediaminetetraacetic acid tubes and kept on ice until separation. Plasma was separated sequentially up to 24 h after sampling and dFdC and dFdU were quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS). The change in plasma concentrations over time was compared with the highest imprecision for concentrations above the lower limit of quantification of the LC-MS/MS method. Analyte concentrations decreased slightly over time, but for samples stored for 4 h on ice, the decline was smaller than the expected analytical imprecision. After 24 h, the maximum decline was 14.0%, which exceeded the expected analytical imprecision. dFdC and dFdU stabilities were acceptable for at least 4 h when THU-spiked whole blood samples were kept on ice. This is within the scope of routine sampling procedures. Further, variations in separation time intervals within this time frame are negligible when interpreting drug concentrations.

Locked 2'-Deoxy-2',4'-Difluororibo Modified Nucleic Acids: Thermal Stability, Structural Studies, and siRNA Activity.

2'-Deoxy-2',4'-difluorouridine (2',4'-diF-rU) was readily incorporated into DNA and RNA oligonucleotides via standard solid phase synthesis protocols. NMR and thermal denaturation (Tm) data of duplexes was consistent with the 2',4'-diF-rU nucleotides adopting a rigid North (RNA-like) sugar conformation, as previously observed for the nucleoside monomer. The impact of this modification on Tm is neutral when incorporated within RNA:RNA duplexes, mildly destabilizing when located in the RNA strand of a DNA:RNA duplex, and highly destabilizing when inserted in the DNA strand of DNA:RNA and DNA:DNA duplexes. Molecular dynamics calculations suggest that the destabilization effect in DNA:DNA and DNA:RNA duplexes is the result of structural distortions created by A/B junctions within the helical structures. Quantum mechanics calculations suggest that the "neutral" effect imparted to A-form duplexes is caused by alterations in charge distribution that compensate the stabilizing effect expected for a pure North-puckered furanose sugar. 2',4'-diF-RNA modified siRNAs were able to trigger RNA interference with excellent efficiency. Of note, incorporation of a few 2',4'-diF-rU residues in the middle of the guide (antisense) strand afforded siRNAs that were more potent than the corresponding siRNAs containing LNA and 2'-F-ANA modifications, and as active as the 2'-F-RNA modified siRNAs.

Development of an LC-MS/MS assay for the quantitative determination of the intracellular 5-fluorouracil nucleotides responsible for the anticancer effect of 5-fluorouracil.

5-Fluorouracil (5-FU) and its oral prodrug capecitabine are among the most widely used chemotherapeutics. For cytotoxic activity, 5-FU requires cellular uptake and intracellular metabolic activation. Three intracellular formed metabolites are responsible for the antineoplastic effect of 5-FU: 5-fluorouridine 5'-triphosphate (FUTP), 5-fluoro-2'-deoxyuridine 5'-triphosphate (FdUTP) and 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). In this paper, we describe the development of an LC-MS/MS assay for quantification of these active 5-FU nucleotides in peripheral blood mononuclear cells (PBMCs). Because the intracellular 5-FU nucleotide concentrations were very low, maximization of the release from the cell matrix and minimization of interference were critical factors. Therefore, a series of experiments was performed to select the best method for cell lysis and nucleotide extraction. Chromatography was optimized to obtain separation from endogenous nucleotides, and the effect of different cell numbers was examined. The assay was validated for the following concentration ranges in PBMC lysate: 0.488-19.9 nM for FUTP, 1.66-67.7 nM for FdUTP and 0.748-30.7 nM for FdUMP. Accuracies were between -2.2 and 7.0% deviation for all analytes, and the coefficient of variation values were ≤ 4.9%. The assay was successfully applied to quantify 5-FU nucleotides in PBMC samples from patients treated with capecitabine and patients receiving 5-FU intravenously. FUTP amounts up to 3054 fmol/10(6) PBMCs and FdUMP levels up to 169 fmol/10(6) PBMCs were measured. The FdUTP concentrations were below the lower limit of quantification. To our knowledge, this is the first time that 5-FU nucleotides were quantified in cells from patients treated with 5-FU or capecitabine without using a radiolabel.

Inhibition of thymidylate synthase by 2',2'-difluoro-2'-deoxycytidine (Gemcitabine) and its metabolite 2',2'-difluoro-2'-deoxyuridine.

2',2'-Difluoro-2'-deoxycytidine (dFdC, gemcitabine) is a cytidine analogue active against several solid tumor types, such as ovarian, pancreatic and non-small cell lung cancer. The compound has a complex mechanism of action. Because of the structural similarity of one metabolite of dFdC, dFdUMP, with the natural substrate for thymidylate synthase (TS) dUMP, we investigated whether dFdC and its deamination product 2',2'-difluoro-2'-deoxyuridine (dFdU) would inhibit TS. This study was performed using two solid tumor cell lines: the human ovarian carcinoma cell line A2780 and its dFdC-resistant variant AG6000. The specific TS inhibitor Raltitrexed (RTX) was included as a positive control. Using the in situ TS activity assay measuring the intracellular conversion of [5-(3)H]-2'-deoxyuridine or [5-(3)H]-2'-deoxycytidine to dTMP and tritiated water, it was observed that dFdC and dFdU inhibited TS. In A2780 cells after a 4h exposure to 1 µM dFdC tritium release was inhibited by 50% but did not increase after 24h, Inhibition was also observed following dFdU at 100 µM. No effect was observed in the dFdC-resistant cell line AG6000; in this cell line only RTX had an inhibitory effect on TS activity. In the A2780 cell line RTX inhibited TS in a time dependent manner. In addition, DNA specific compounds such as 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentafuranosylcytosine and aphidicoline were utilized to exclude DNA inhibition mediated down regulation of the thymidine kinase. Inhibition of the enzyme resulted in a relative increase of mis-incorporation of [5-(3)H]-2'-deoxyuridine into DNA. In an attempt to elucidate the mechanism of in situ TS inhibition the ternary complex formation and possible inhibition in cellular extracts of A2780 cells, before and after exposure to dFdC, were determined. With the applied methods no proof for formation of a stable complex was found. In simultaneously performed experiments with 5FU such a complex formation could be demonstrated. However, using purified TS it was demonstrated that dFdUMP and not dFdCMP competitively inhibited TS with a Ki of 130 µM, without ternary complex formation. In conclusion, in this paper we reveal a new target of dFdC: thymidylate synthase.

Effect of socioeconomic status on the association between air pollution and mortality in Bogota, Colombia / Efecto del nivel socioeconómico sobre la asociación entre contaminantes atmosféricos y mortalidad en Bogotá, Colombia

Objective. To evaluate the modification effect of socioeconomic status (SES) on the association between acute exposure to particulate matter less than 10 microns in aerodynamic diameter (PM10) and mortality in Bogota, Colombia. Materials and methods. A time-series ecological study was conducted (1998-2006). The localities of the cities were stratified using principal components analysis, creating three levels of aggregation that allowed for the evaluation of the impact of SES on the relationship between mortality and air pollution. Results. For all ages, the change in the mortality risk for all causes was 0.76% (95%CI 0.27-1.26) for SES I (low), 0.58% (95%CI 0.16-1.00) for SES II (mid) and -0.29% (95%CI -1.16-0.57) for SES III (high) per 10µg/m³ increment in the daily average of PM10 on day of death. Conclusions. The results suggest that SES significantly modifies the effect of environmental exposure to PM10 on mortality from all causes and respiratory causes.

Objetivo. Evaluar el efecto modificador del nivel socioeconómico (NSE) sobre la asociación entre la exposición aguda a partículas menores de 10 micras de diámetro aerodinámico (PM10) y la mortalidad en Bogotá, Colombia. Material y métodos. Se realizó un estudio ecológico de series de tiempo (1998-2006). Mediante análisis de componentes principales se estableció una estratificación de las localidades de la ciudad, de lo que se generaron tres niveles de agregación que permitieron evaluar el impacto de la variable NSE en la relación mortalidad-contaminación atmosférica. Resultados. En todas las edades, para la mortalidad por todas las causas, el porcentaje de cambio en el riesgo fue 0.76% (IC95% 0.27-1.26) en el NSE I (bajo), 0.58% (IC95% 0.16-1.00) en el NSE II (medio) y -0.29% (IC95% -1.16-0.57) en el NSE III (alto), por incremento de 10µg/m³ en el promedio diario de PM10 en el día del deceso. Conclusiones. Los resultados sugieren que el NSE modifica de manera significativa el efecto de la exposición ambiental a PM10 sobre la mortalidad por todas las causas y causas respiratorias.

Rigid 2',4'-difluororibonucleosides: synthesis, conformational analysis, and incorporation into nascent RNA by HCV polymerase.

We report on the synthesis and conformational properties of 2'-deoxy-2',4'-difluorouridine (2',4'-diF-rU) and cytidine (2',4'-diF-rC) nucleosides. NMR analysis and quantum mechanical calculations show that the strong stereoelectronic effects induced by the two fluorines essentially "lock" the conformation of the sugar in the North region of the pseudorotational cycle. Our studies also demonstrate that NS5B HCV RNA polymerase was able to accommodate 2',4'-diF-rU 5'-triphosphate (2',4'-diF-rUTP) and to link the monophosphate to the RNA primer strand. 2',4'-diF-rUTP inhibited RNA synthesis in dinucleotide-primed reactions, although with relatively high half-maximal inhibitory concentrations (IC50 > 50 µM). 2',4'-diF-rU/C represents rare examples of "locked" ribonucleoside mimics that lack a bicyclic ring structure.

Synthesis of 3'-azido-2',3'-dideoxy-5-fluorouridine phosphoramidates and evaluation of their anticancer activity.

A series of novel 4-chlorophenyl N-alkyl phosphoramidates of 3'-azido-2',3'-dideoxy-5-fluorouridine (12-21) were synthesized by means of phosphorylation of 3'-azido-2',3'-dideoxy-5-fluorouridine (4) with 4-chlorophenyl phosphoroditriazolide (10) followed by a reaction with the appropriate amine. The synthesized phosphoramidates (12-21) were evaluated for their cytotoxic activity in three human cancer cell lines: cervical (HeLa), oral (KB) and breast (MCF-7) using the sulforhodamine B (SRB) assay. The highest activity in all the investigated cancer cells was displayed by phosphoramidate 13 with the N-ethyl substituent and its activity was much higher than that of the parent nucleoside. Also phosphoramidate 17 with the N-propargyl substituent exhibited good activity in all the used cell lines.

UDP-glucuronosyltransferase (UGT) 1A1*28 polymorphism-directed phase II study of irinotecan with 5'-deoxy-5-fluorouridine (5'-DFUR) for metastatic colorectal cancer.

AIM: We performed a phase II study of irinotecan with 5'-deoxy-5-fluorouridine (5'-DFUR) for metastatic colorectal cancer based on UDP-glucuronosyltransferase (UGT) 1A1 polymorphism. PATIENTS AND METHODS: A total of 28 patients were enrolled. The dose of irinotecan was 150 mg/m(2) for patients with the *1/*1 wild-type genotype, and 70 mg/m(2) for those with the *1/*28 mutated genotype. The primary end-point was the response rate (RR); secondary end-points were safety, time to treatment failure (TTF), and overall survival (OS). RESULTS: In 28 patients total, genotype was wild-type in 22 and mutated in six. The RR was *1/*1 (22.7%; wild-type) vs. *1/*28 (16.7%; mutated); the median TTF was 5 months vs. 4.5 months, and the median OS was 13 months vs. 17.5 months, respectively. None of these differences were significant. Toxicities of grade 3 or higher were neutropenia (9.0% vs. 0%, respectively) and diarrhea (13.6% vs. 0%, respectively). CONCLUSION: This genotype-oriented therapy was effective and safe, and thus appears useful for patients who have complications or advanced age.