Assessment of efficacy of postinfusion tubing flushing in reducing risk of cytotoxic contamination.

PURPOSE: Infusion of cytotoxic drugs carries the risk of occupational exposure of healthcare workers. Since disconnecting an infusion line is a source of contamination, flushing of tubing after infusion of cytotoxic agents is recommended, but the optimal volume of rinsing solution is unknown. The objective of this study was to assess whether postinfusion line flushing completely eliminates cytotoxics. METHODS: Infusions were simulated with 3 cytotoxics (gemcitabine, cytarabine, and paclitaxel) diluted in 5% dextrose injection or 0.9% sodium chloride injection in 250-mL infusion bags. Infusion lines were flushed using 5% dextrose injection or 0.9% sodium chloride solution at 2 different flow rates. The remaining concentration of cytotoxics in the infusion line was measured by a validated high-performance liquid chromatography (HPLC) method after passage of every 10 mL of flushing volume until a total of 100 mL had been flushed through. RESULTS: All cytotoxics remained detectable even after line flushing with 80 mL of flushing solution (a volume 3-fold greater than the dead space volume within the infusion set). Gemcitabine and cytarabine were still quantifiable via HPLC even after flushing with 100 mL of solution. Efficacy of flushing was influenced by the lipophilicity of drugs but not by either the flushing solvent used or the flushing flow rate. After 2-fold dead space volume flushing, the estimated amount of drug remaining in the infusion set was within 0.19% to 0.56% of the prescribed dose for all 3 cytotoxics evaluated. CONCLUSION: Complete elimination of cytotoxics from an infusion line is an unrealistic objective. Two-fold dead space volume flushing could be considered optimal in terms of administered dose but not from an environmental contamination point of view. Even when flushed, the infusion set should still be considered a source of cytotoxic contamination.

Electrochemical molecularly bioimprinted siloxane biosensor on the basis of core/shell silver nanoparticles/EGFR exon 21 L858R point mutant gene/siloxane film for ultra-sensing of Gemcitabine as a lung cancer chemotherapy medication.

In search for improvements in bioanalysis electrochemical sensors, for better assessment of anti-cancer drugs, it is necessary for their detection limits to be minimized and the sensitivity and selectivity to be surpassed simultaneously; whereas, resolving any probable interfering with other medical treatments are considered. In this work, a novel approach was adopted for detection and assessment of Gemcitabine (GEM) as an anti-cancer drug based on evaluating its interaction with EGFR exon 21-point mutant gene. An electrochemical nanobiosensor was invented based on a new molecularly bioimprinted siloxane polymer (MBIS) strategy; in which the EGFR exon 21 acts as an identification probe. The roles of multi-walled carbon nanotubes and Ag nanoparticles (NPs) are to perform as a signal amplifier. The MBIS film was prepared by acid-catalysed hydrolysis/condensation of the sample solution, containing Ag NPs, ds-DNA of EGFR exon 21 point mutant gene, GEM as a template molecule, 3-(aminopropyl) trimethoxysilane (APTMS) and tetraethoxysilane. The interaction between the dsDNA and GEM was investigated by employing the modified biosensor and monitoring oxidation signal of guanine and adenine. The produced biosensor was characterized by XRD, FE-SEM, EDS, FT-IR and differential pulse voltammetry. The oxidation signals of adenine and guanine were in linear range when the device was subjected to various concentrations of GEM, from 1.5 to -93 µM, where a low detection limit 12.5 nmol L-1, and 48.8 nmol L-1 were recorded by guanine and adenine respectively. The developed biosensor did perform very well when employed for the actual samples; the stability was also approved which was acceptable for a reasonable time.

Enrichment and Quantitative Determination of 5-(Hydroxymethyl)-2'-deoxycytidine, 5-(Formyl)-2'-deoxycytidine, and 5-(Carboxyl)-2'-deoxycytidine in Human Urine of Breast Cancer Patients by Magnetic Hyper-Cross-Linked Microporous Polymers Based on Polyionic Liquid.

5-(Hydroxymethyl)-2'-deoxycytidine (5-hmdC), 5-(formyl)-2'-deoxycytidine (5-fodC), and 5-(carboxyl)-2'-deoxycytidine (5-cadC) are crucial intermediate products of the DNA demethylation pathway, which can also act as potential biomarkers reflecting the diagnosis and prognosis in multiple tumors. Detecting 5-hmdC, 5-fodC, and 5-cadC in human urine has various advantages including readily available samples and being noninvasive to patients. However, few works have reported the detection of 5-fodC and 5-cadC due to their trace amounts. Here we developed a novel magnetic hyper-cross-linked microporous polymer (HMP) material based on polyionic liquid (PIL) for the enrichment of 5-hmdC, 5-fodC, and 5-cadC. These magnetic PIL-HMP materials provided specific enrichment superiority for three modified cytidines. After enrichment, the signal intensity of 5-hmdC, 5-fodC, and 5-cadC increased 10-75-fold with lower limits of quantitation (LLOQ) of 0.049, 0.781, and 0.781 ng/mL, respectively. The recoveries were approximately 86.5-95.2% for 5-hmdC, 95.2-107.8% for 5-fodC, and 99.4-102.4% for 5-cadC under the relative standard deviation (RSD) of 0.2-10.3%. Finally, we successfully applied magnetic PIL-HMP materials coupled with high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) in enrichment and quantitative determination of 5-hmdC, 5-fodC, and 5-cadC in human urine of 10 breast cancer patients and 10 healthy people. We found that the level of 5-hmdC decreased in breast cancer patients ( p < 0.05), while the levels of 5-fodC and 5-cadC increased ( p < 0.05, p < 0.01). Our results demonstrated that the levels of metabolic 5-hmdC, 5-fodC, and 5-cadC in human urine are closely related to breast cancer, which could contribute to the clinical diagnosis and investigation of breast cancer and its occurrence and development mechanisms.

Fluorescent Immortalized Human Adipose Derived Stromal Cells (hASCs-TS/GFP+) for Studying Cell Drug Delivery Mediated by Microvesicles.

BACKGROUND: A new tool for the drug delivery is based on the use of Mesenchymal Stromal Cells (MSCs) loaded in vitro with anti-cancer drugs. Unfortunately, the restricted lifespan of MSCs represents a significant limitation to produce them in high amounts and for long time studies. Immortalized MSCs from adipose tissue (hASCs) have been generated as good source of cells with stable features. These cells could improve the development of standardized procedures for both in vitro and preclinical studies. Furthermore they facilitate procedures for preparing large amounts of secretome containing microvesicles (MVs). METHOD: We used human adipose tissue derived MSCs immortalized with hTERT+SV40 (TS) genes and transfected with GFP (hASCs-TS/GFP+). This line was investigated for its ability to uptake and release anticancer drugs. Microvesicles associated to paclitaxel (MVs/PTX) were isolated, quantified, and tested on pancreatic cancer cells. RESULTS: The line hASCs-TS/GFP+ maintained the main mesenchymal characters and was able to uptake and release, in active form, both paclitaxel and gemcitabine. From paclitaxel loaded hASCs-TS/GFP+ cells were isolated microvesicles in sufficient amount to inhibit "in vitro" the proliferation of pancreatic tumor cells. CONCLUSION: Our study suggests that human immortalized MSCs could be used for a large scale production of cells for mediated drug delivery. Moreover, the secretion of drug-associated MVs could represent a new way for producing new drug formulation by "biogenesis". In the context of the "advanced cell therapy procedure", the MVs/PTX production would use less resource and time and it could possibly contribute to simplification of GMP procedures.

An evaluation of Minor Groove Binders as anti-lung cancer therapeutics.

A series of 47 structurally diverse MGBs, derived from the natural product distamycin, was evaluated for anti-lung cancer activity by screening against the melanoma cancer cell line B16-F10. Five compounds have been found to possess significant activity, more so than a standard therapy, Gemcitabine. Moreover, one compound has been found to have an activity around 70-fold that of Gemcitabine and has a favourable selectivity index of greater than 125. Furthermore, initial studies have revealed this compound to be metabolically stable and thus it represents a lead for further optimisation towards a novel treatment for lung cancer.

Optimization of global DNA methylation measurement by LC-MS/MS and its application in lung cancer patients.

Global analyses of DNA methylation contribute important insights into biology and the wide-ranging role of DNA methylation. We describe the use of online solid-phase extraction and isotope-dilution liquid chromatography/tandem mass spectrometry (LC-MS/MS) for the simultaneous measurement of 5-methyl-2'-deoxycytidine (5-medC) and 2'-deoxycytidine (dC) in DNA. With the incorporation of isotope internal standards and online enrichment techniques, the detection limit of this method was estimated to be as low as 0.065 pg which enables human global DNA methylation detection using only picogram amounts of DNA. This method was applied to assess the optimal amounts of enzymes required for DNA digestion regarding an accurate global DNA methylation determination and completeness of digestion and to determine global methylation in human tumor adjacent lung tissue of 79 lung cancer patients. We further determined methylated (N7-methylguanine (N7-meG), O (6)-methylguanine (O (6)-meG), and N3-methyladenine (N3-meA)) and oxidized DNA lesions (8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG)) in lung cancer patients by LC-MS/MS. Optimization experiments revealed that dC was liberated from DNA much more readily than 5-medC by nuclease P1 and alkaline phosphatase (AP) in DNA, which could lead to an error in the global DNA methylation measurement following digestion with insufficient enzymes. Nuclease P1 showed more differential activity for 5-medC and dC than AP. Global DNA methylation levels in adenocarcinoma and squamous cell carcinoma patients were similar in the range of 3.16-4.01 %. Global DNA methylation levels were not affected by smoking and gender and were not correlated with N7-meG or 8-oxodG in lung cancer patients. Levels of O (6)-meG and N3-meA were however found to be undetectable in all lung tissue samples.

Synthesis and evaluation of molecularly imprinted polymeric microspheres for highly selective extraction of an anti-AIDS drug emtricitabine.

The molecularly imprinted polymeric microspheres (MIPMs, 3~5 µm), used as high-performance liquid chromatography (HPLC) and solid-phase extraction (SPE) packing materials for anti-AIDS drug emtricitabine (FTC), were synthesized by precipitation polymerization. The effects of ratio of chloroform to acetonitrile on the morphology and diameter of MIPMs were investigated. The prepared MIPMs were characterized by HPLC. The imprinting factor (2.26) suggests that the resultant MIPMs exhibit good recognition and affinity to FTC. In addition, the MIPMs were used in SPE as packing material for separation and enrichment of FTC. The recovery of FTC on MIPMs cartridge was 97.6 % in standard solution. Finally, the MIPMs cartridge was applied to extract the FTC in human serum samples. Impurities in sample have been mostly removed, and the average recovery of 92.5 % was obtained with a detection limit of 0.005 µg/mL and a linear range of 0.02~4.0 µg/mL. The method established can be used to monitor the FTC in human serum sample with good accuracy and selectivity.

Determination of nucleoside analog mono-, di-, and tri-phosphates in cellular matrix by solid phase extraction and ultra-sensitive LC-MS/MS detection.

An ultra-sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed and validated to facilitate the assessment of clinical pharmacokinetics of nucleotide analogs from lysed intracellular matrix. The method utilized a strong anion exchange isolation of mono-(MP), di-(DP), and tri-phosphates (TP) from intracellular matrix. Each fraction was then dephosphorylated to the parent moiety yielding a molar equivalent to the original nucleotide analog intracellular concentration. The analytical portion of the methodology was optimized in specific nucleoside analog centric modes (i.e. tenofovir (TFV) centric, zidovudine (ZDV) centric), which included desalting/concentration by solid phase extraction and detection by LC-MS/MS. Nucleotide analog MP-, DP-, and TP-determined on the TFV centric mode of analysis include TFV, lamivudine (3TC), and emtricitibine (FTC). The quantifiable linear range for TFV was 2.5-2000 fmol/sample, and that for 3TC/FTC was 0.1 200 pmol/sample. Nucleoside analog MP-, DP-, and TP-determined on the ZDV centric mode of analysis included 3TC and ZDV. The quantifiable linear range for 3TC was 0.1 100 pmol/sample, and 5-2000 fmol/sample for ZDV. Stable labeled isotopic internal standards facilitated accuracy and precision in alternative cell matrices, which supported the intended use of the method for MP, DP, and TP determinations in various cell types. The method was successfully applied to clinical research samples generating novel intracellular information for TFV, FTC, ZDV, and 3TC nucleotides. This document outlines method development, validation, and application to clinical research.

Fused-core silica column ultra-performance liquid chromatography-ion trap tandem mass spectrometry for determination of global DNA methylation status.

Epigenetic modifications, such as DNA methylation, play key roles in transcriptional regulation of gene expression. More recently, global DNA methylation levels have been documented to be altered in several diseases, including cancer, and as the result of exposure to environmental toxicants. Based on the potential use of global DNA methylation status as a biomarker of disease status and exposure to environmental toxicants, we sought to develop a rapid, sensitive, and precise analytical method for the quantitative measurement of global DNA methylation status using ultra-performance liquid chromatography with detection by ion trap tandem mass spectrometry. Using a fused-core silica column, 2'-deoxyguanosine (2dG) and 5-methyl-2'-deoxycytidine (5mdC) were resolved in less than 1 min with detection limits of 0.54 and 1.47 fmol for 5mdC and 2dG, respectively. The accuracy of detection was 95% or higher, and the day-to-day coefficient of variation was found to be 3.8%. The method was validated by quantification of global DNA methylation status following treatment of cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine, which reduced DNA methylation from 3.1% in control cells to 1.1% in treated cells. The sensitivity and high throughput of this method rend it suitable for large-scale analysis of epidemiological and clinical DNA samples.

Simultaneous quantification of 2',2'-difluorodeoxycytidine and 2',2'-difluorodeoxyuridine nucleosides and nucleotides in white blood cells using porous graphitic carbon chromatography coupled with tandem mass spectrometry.

A novel assay for the simultaneous quantification of the widely used anticancer agent 2',2'-difluorodeoxycytidine (gemcitabine; dFdC), its deaminated metabolite 2',2'-difluorodeoxyuridine (dFdU) and their mono-, di- and triphosphates (dFdCMP, dFdCDP, dFdCTP, dFdUMP, dFdUDP and dFdUTP) in peripheral blood mononuclear cells (PBMCs) is described. Separation of all eight compounds was achieved within 15 min using a porous graphitic carbon column (Hypercarb) with a gradient from 0 to 25 mM ammonium bicarbonate in acetonitrile/water (15:85, v/v). Calibration ranges in PBMC lysate from 4.29 to 429, 29.0 to 2900, 31.4 to 3140 and 36.9 to 3690 nM for dFdC, dFdCMP, dFdCDP and dFdCTP and from 42.1 to 4210, 25.4 to 2540, 43.2 to 4320 and 52.7 to 5270 nM for dFdU, dFdUMP, dFdUDP and dFdUTP, respectively, were validated. Accuracies were within 82.3-119% at the lower limit of quantification (LLOQ) and the precisions were less than 20.0%. At the other tested levels accuracies were within 91.4-114% and precisions less than 14.9%. Mixtures of (13)C,(15)N(2)-labeled dFdC and dFdU nucleotides were synthesized and used as internal standards. Whole blood samples showed extensive ongoing dFdC metabolism when stored at room temperature, but not on ice-water, which made the addition of enzyme inhibitors unnecessary. Stock solutions and samples were stable under all analytically relevant conditions. The method was successfully applied to clinical samples.

High-performance capillary electrophoretic method for the quantification of global DNA methylation: application to methotrexate-resistant cells.

Global DNA hypomethylation in tumor tissue is a common characteristic in a variety of malignancies such as breast, colon, oral, lung, and blood cancers. A rapid and sensitive method has been developed for the determination of global DNA methylation in cells. Five substances--2'-deoxycytidine (dC), 5-methyl 2'-deoxycytidine (mdC), 2'-deoxyadenosine (dA), 2'-deoxythymidine (dT), and 2'-deoxyguanosine (dG)--were completely separated by high-performance capillary electrophoresis in 10 min. Intraday coefficient of variation was less than 1%, and interday coefficient of variation was less than 2%. The minimal detection limit was 1 microM. Acquired drug resistance to methotrexate (MTX) is one of the most serious problems in cancer chemotherapy. Under optimal conditions, we analyzed global DNA methylation levels in A549 and A549/MTX cells, and only 10(5) cells are needed to obtain reliable results. The percentage of 5-methyl-2'-deoxycytidine (5-mC) was 4.80+/-0.52% in A549 cells, and this decreased to 4.20+/-0.44% in A549/MTX cells. It was considered as statistically significant. This demonstrated that the mechanisms of acquired drug resistance to MTX might be concerned with DNA methylation.

Comparison of two column characterisation systems based on pharmaceutical applications.

A useful column characterisation system should help chromatographers to select the most appropriate column to use, e.g. when a particular chromatographic column is not available or when facing the dilemma of selecting a suitable column for analysis according to an official monograph. Official monographs of the European Pharmacopoeia and the United States Pharmacopeia are not allowed to mention the brand name of the stationary phase used for the method development. Also given the overwhelming offer of several hundreds of commercially available reversed-phase liquid chromatographic columns, the choice of a suitable column could be difficult sometimes. To support rational column selection, a column characterisation study was started in our laboratory in 2000. In the same period, Euerby et al. also developed a column characterisation system, which is now released as Column Selector by ACD/Labs. The aim of this project was to compare the two existing column characterisation systems, i.e. the KUL system and the Euerby system. Other research groups active in this field will not be discussed here. Euerby et al. developed a column characterisation system based on 6 test parameters, while the KUL system is based on 4 chromatographic parameters. Comparison was done using a set of 63 columns. For 7 different pharmaceutical separations (fluoxetine, gemcitabine, erythromycin, tetracycline, tetracaine, amlodipine and bisacodyl), a ranking was built based on an F-value (KUL method) or Column Difference Factor value (Euerby method) versus a (virtual) reference column. Both methods showed a similar ranking. The KUL and Euerby methods do not perfectly match, but they yield very similar results, allowing with a relatively high certainty, the selection of similar or dissimilar columns as compared to a reference column. An analyst that uses either of the two methods, will end up with a similar ranking. From a practical point of view, it must be noted that the KUL method only includes 4 parameters and 3 chromatographic methods compared to 6 parameters and 4 methods for the Euerby method. Hence, the time needed to determine the chromatographic properties of a column is shorter for the KUL approach. Access to the KUL method also requires no download procedures.

Isolation of an unknown metabolite of capecitabine, an oral 5-fluorouracil prodrug, and its identification by nuclear magnetic resonance and liquid chromatography-tandem mass spectrometry as a glucuroconjugate of 5'-deoxy-5-fluorocytidine, namely 2'-(beta-D-glucuronic acid)-5'-deoxy-5-fluorocytidine.

A new metabolite of capecitabine, a prodrug of 5-fluorouracil, was detected by (19)F NMR in bile and liver of rats treated with this anticancer drug. Crude bile and perchloric acid extract of liver was subjected to liquid-liquid separation followed by a pre-purification step on a preparative octadecyl silane column (C(18)). The compound was purified by HPLC optimised to allow the detection of the unknown metabolite and its assumed precursor 5'-deoxy-5-fluorocytidine (5'-DFCR). Treatment with beta-glucuronidase from three sources showed that it was a glucuroconjugate of 5'-DFCR. HPLC-TIS-MS-MS and (1)H NMR allowed identification of the unknown metabolite as 2'-(beta-D-glucuronic acid)-5'-deoxy-5-fluorocytidine.

Comparison of the isolation of adducts of 2'-deoxycytidine and 2'-deoxyguanosine with phenylglycidyl ether by high-performance liquid chromatography on a reversed-phase column and a polystyrene-divinylbenzene column.

2'-Deoxycitidine (dCyd) and 2'-deoxyguanosine (dGuo) were subjected to reaction with phenylglycidyl ether (PGE) in methanol in order to study the formation of the corresponding 2'-deoxynucleoside adducts. Separation methods were developed on analytical and semi-preparative scales using high-performance liquid chromatography with photodiode-array detection on a reversed-phase column and on a polystyrene-divinylbenzene column. The use of the latter column was prompted by decomposition of the preparatively isolated dGuo-PGE adducts on the reversed-phase column. The use of a polystyrene-divinylbenzene column solved this problem and also revealed the presence of one more peak in both the dCyd- and dGuo-PGE reaction mixtures. The adducts of dCyd and dGuo were isolated on preparative reversed-phase and polystyrene-divinylbenzene columns and characterized by UV, fast atom bombardment mass and 360 MHz 1H NMR spectrometry. The adducts of dCyd were the diastereomers of N-3-(2-hydroxy-3-phenoxypropyl)-2'-deoxycytidine and N4-(2-hydroxy-3-phenoxypropyl)-2'-deoxycytidine whereas those of dGuo were the two diastereomers of N-7-(2-hydroxy-3-phenoxypropyl)-2'-deoxyguanosine and a third peak which appeared to be mainly N2-(2-hydroxy-3-phenoxypropyl)-2'-deoxyguanosine.

Exposure of JB-6 mouse epidermal cells to 12-O-tetradecanoyl-phorbol-13-acetate is not accompanied by a significant change in total DNA-cytosine methylation.

The extent of methylation of the cytosine bases in DNA is believed to be a major factor influencing gene expression in eukaryotic cells. We have asked whether the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) alters the amount of 5-methylcytosine in DNA. The amount and relative distribution of 5-methylcytosine in the DNA of two subclones of the JB-6 mouse epidermal cell line were determined respectively by high performance liquid chromatography and digestion with the restriction enzymes MspI and HpaII. Exposure to TPA for up to several cell generations had no detectable effect on the degree of DNA methylation (3.9% of the total cytosine) in the two JB-6 lines or Friend erythroleukemia cells. Reduced methylation was readily detected in DNA extracted from cells exposed to 5-azacytidine. The data suggest that tumor promotion (at least that induced by TPA) is likely not the consequence of a generalized elevation or reduction in the amount of 5-methyl-cytosine in the DNA.