Development of LC-HRMS methods for evaluation of metabolic conversion of 5-fluorocytosine at GDEPT procedure.

Gene-directed enzyme/prodrug therapy represents one of the experimental treatment approaches. The system based on conversion of nontoxic prodrug 5-fluorocytosine to chemotherapeutic 5-fluorouracil by cytosine deaminase or fusion cytosine deaminase::uracil phosphoribosyl transferase belongs to the most frequently used. The detailed analysis of 5-fluorocytosine, 5-fluorouracil and its metabolites enables to understand various responses of tumour cells to treatment as well as mechanisms of resistance. A fast, sensitive and accurate methods based on liquid chromatography with high-resolution mass spectrometry (LC-HRMS) for the identification and quantification of 5-fluorocytosine, 5-fluorouracil and its major metabolites were developed. Two different hybrid high-resolution mass spectrometers sufficient for study of metabolic pathways were used. The LC-ESI IT-TOF MS method was successfully used for identification of 5-fluorocytosine, 5-fluorouracil and its metabolites in complex biological matrices (mesenchymal stromal cells and tumour cells media) and for confirmation of the metabolic conversion of 5-fluorocytosine even in chemoresistant tumour cells media samples. For quantification, the LC-HESI QExactive MS method was developed and validated. The developed method demonstrated a very good linear range for 5-fluorocytosine from 1 ng/mL to 1000 ng/mL and for its major metabolites from 5 ng/mL to 1000 ng/mL. The limits of detection and limits of quantification ranged from 1.1 to 26 ng/mL and from 3.6 to 87 ng/mL, respectively. Both developed methods confirmed the ability of gene-directed enzyme prodrug therapy to metabolically convert 5-fluorocytosine to 5-fluorouracil and its major metabolites in real samples of tumour cell media and mesenchymal stromal cells.

Determination of 5-fluorocytosine, 5-fluorouracil, and 5-fluorouridine in hospital wastewater by liquid chromatography-mass spectrometry.

Chemotherapeutics are pharmaceutical compounds the occurrence of which in the environment is of growing concern because of the increase in treatments against cancer diseases. They can reach the aquatic ecosystems after passing through wastewater treatment plants without complete removal. One of the most frequently used chemotherapeutics is 5-fluorouracil which exhibits a strong cytostatic effect. In this paper, an analytical methodology was developed, validated, and applied to determine 5-fluorouracil, its precursor, 5-fluorocytosine, and its major active metabolite, 5-fluorouridine, in hospital wastewater samples. Due to the expected low concentrations after dilution and interferences present in such a complex matrix, a very selective and sensitive detection method is required. Moreover, an extraction method must be implemented prior to the determination in order to purify the sample extract and preconcentrate the target analytes at micrograms per liter concentration levels. Solid-phase extraction followed by liquid chromatography with tandem mass spectrometry was the combination of choice and all included parameters were studied. Under optimized conditions for wastewater samples analysis, recoveries from 63 to 108% were obtained, while intraday and interday relative standard deviations never exceeded 20 and 25%, respectively. Limits of detection between 61 and 620 ng/L were achieved. Finally, the optimized method was applied to samples from hospital wastewater effluents.

Cytotoxic response of 5-fluorouracil-resistant cells to gene- and cell-directed enzyme/prodrug treatment.

Gene-directed enzyme/prodrug therapy (GDEPT) mediated by mesenchymal stromal cells (MSC) was already approved for clinical study on a progressive disease refractory to standard therapy. In this work, we examined the effect of several GDEPT approaches on chemoresistant cells. First, we derived 5-fluorouracil (5-FU)-resistant variant of human colorectal adenocarcinoma cells HT-29 designated HT-29/EGFP/FUR. Our data show that the upregulation of thymidylate synthase (TS) and downregulation of thymidine phosphorylase (TP), orotate phosphoribosyl transferase (OPRT) and dihydropyrimidine dehydrogenase (DPD) contributed to the 5-FU resistance in cancer cells. Next, we combined the MSC expressing either yeast cytosine deaminase (CD-MSC) or fusion yeast CD::uracil phosphoribosyl transferase (CD::UPRT-MSC) and prodrug 5-fluorocytosine (5-FC) in a cell-mediated GDEPT approach. Bystander cytotoxic effect in the direct co-cultures of the tumor and therapeutic cells mixed in a 5:1 ratio resulted in 55% and 70% inhibition of proliferation, respectively. However, the acquired chemoresistance to 5-FU can be overcome by introducing the prodrug-converting transgene into the tumor cells. When the transgene CD::UPRT was expressed in the chemoresistant cells (CD::UPRT-FUR), substantial suicide effect and a 90% decrease in viability was observed using non-toxic concentration of 62.5 µg/ml 5-FC. In summary, we demonstrate here that the transgene introduction circumvented 5-FU resistance in the tumor cells.