Synergistic Antitumor Effect of 5-Fluorouracil Combined with Constituents from Pleurospermum lindleyanum in Hepatocellular Carcinoma SMMC-7721 Cells.

BACKGROUND: A Chinese folk medicine plant Pleurospermum lindleyanum possesses pharmacological activities of heat-clearing, detoxifying and preventing from hepatopathy, coronary heart disease, hypertension, and high altitude sickness. We isolated and characterized its constituents to investigate its synergistic effects against human hepatoma SMMC-7721 cells. OBJECTIVE: The aim of this study was to explore the synergistic anti-cancer activities of isolates from P. lindleyanum with 5-FU on hepatoma SMMC-7721 cells in vitro and their primary mechanisms. METHODS: Sequential chromatographic techniques were conducted for the isolation studies. The isolate's structures were established by spectroscopic analysis as well as X-ray crystallographic diffraction. Growth inhibition was detected by MTT assay. The isobologram method was used to assess the effect of drug combinations. Flow cytometry and western blot were used to examine apoptosis and protein expression. RESULTS: A new coumarin (16), along with sixteen known compounds, were isolated from the whole plant of P. lindleyanum and their structures were elucidated by spectroscopic methods. Four coumarins (2, 3, 5, and 16), two flavonoids (8 and 9) and three phytosterols and triterpenes (12-14) were found to synergistically enhance the inhibitory effect of 5-FU against SMMC-7721 cells. Among them, compounds 3 and 16 exhibited the best synergistic effects with IC50 of 5-FU reduced by 16-fold and 22-fold possessing the minimum Combination Index (CI) 0.34 and 0.27. The mechanism of action of combinations might be through synergistic arresting for the cell cycle at G1 phases and the induction of apoptosis. Moreover, western blotting and molecular docking revealed that compounds 3 or 5 might promote 5-FU-induced apoptosis by regulating the expression of Caspase 9 and PARP. CONCLUSION: Constituents from P. lindleyanum may improve the treatment effectiveness of 5-FU against hepatocellular carcinoma cells.

Photocatalytical removal of fluorouracil using TiO2-P25 and N/S doped TiO2 catalysts: A kinetic and mechanistic study.

In the present study, the photocatalytic activity of TiO2-based photocatalysts toward degradation and mineralization of the anti-cancer drug 5-fluorouracil (5-FU) in aqueous phase was investigated under simulated solar and visible irradiation. Commercial TiO2 (P25) and N/S-doped TiO2 catalysts synthesized by a simple sol-gel method were used as photocatalysts. TiO2 P-25 was found to be the most photoactive catalyst for the removal of 5-FU, under simulated solar irradiation. Among N/S-doped TiO2 catalysts, the one with molar Ti:N/S ratio equal to 0.5 was the most efficient under simulated solar irradiation. In contrast, under visible irradiation the catalyst with equimolar Ti:N/S ratio showed the highest performance for the removal of 5-FU. Scavenging experiments revealed that HO radicals and h+ were the major reactive species mediating photocatalytic degradation of 5-FU using TiO2 P-25 and N/S-doped TiO2 catalysts, under simulated solar irradiation. On the other hand, the essential contribution of 1O2 and O2- in the degradation of 5-FU under visible light was proved. The transformation products (TPs) of 5-FU, were identified by LC-MS-TOF suggesting that defluorination followed by hydroxylation and oxidation are the main transformation pathways, under all the studied photocatalytic systems.

Removal of highly polar micropollutants from wastewater by powdered activated carbon.

Due to concerns about ecotoxicological effects of pharmaceuticals and other micropollutants released from wastewater treatment plants, activated carbon adsorption is one of the few processes to effectively reduce the concentrations of micropollutants in wastewater. Although aimed mainly at apolar compounds, polar compounds are also simultaneously removed to a certain extent, which has rarely been studied before. In this study, adsorption isotherm and batch kinetic data were collected with two powdered activated carbons (PACs) to assess the removal of the polar pharmaceuticals 5-fluorouracil (5-Fu) and cytarabine (CytR) from ultrapure water and wastewater treatment plant effluent. At pH 7.8, single-solute adsorption isotherm data for the weak acid 5-Fu and the weak base CytR showed that their adsorption capacities were about 1 order of magnitude lower than those of the less polar endocrine disrupting chemicals bisphenol A (BPA) and 17-α-ethinylestradiol (EE2). To remove 90 % of the adsorbate from a single-solute solution 14, 18, 70, and 87 mg L(-1) of HOK Super is required for EE2, BPA, CytR, and 5-Fu, respectively. Effects of solution pH, ionic strength, temperature, and effluent organic matter (EfOM) on 5-Fu and CytR adsorption were evaluated for one PAC. Among the studied factors, the presence of EfOM had the highest effect, due to a strong competition on 5-Fu and CytR adsorption. Adsorption isotherm and kinetic data and their modeling with a homogeneous surface diffusion model showed that removal percentage in the presence of EfOM was independent on the initial concentration of the ionizable compounds 5-Fu and CytR. These results are similar to neutral organic compounds in the presence of natural organic matter. Overall, results showed that PAC doses sufficient to remove >90 % of apolar adsorbates were able to remove no more than 50 % of the polar adsorbates 5-Fu and CytR and that the contact time is a critical parameter.

Preparation of 5-fluorouracil nanoparticles by supercritical antisolvents for pulmonary delivery.

This study concerns the supercritical antisolvent process which allows single-step production of 5-fluorouracil (5-FU) nanoparticles. This process enhances the physical characteristics of 5-FU in order to deliver it directly to the respiratory tract. Several mixtures of methanol with dichloromethane, acetone, or ethanol were used for particle preparation, and their effects on the physical characteristics of the final products were studied. The conditions of the experiment included pressures of 100 and 150 bar, temperature of 40°C, and a flow rate of 1 mL/min. The particles were characterized physicochemically before and after the process for their morphology and crystallinity. In spite of differences in size, the particles were not very different regarding their morphology. The resulting particles were of a regular shape, partly spherical, and appeared to have a smooth surface, whereas the mechanically milled particles showed less uniformity, had surface irregularities and a high particle size distribution, and seemed aggregated. Particles of 5-FU precipitated from methanol-dichloromethane 50:50 had a mean particle size of 248 nm. In order to evaluate the aerodynamic behavior of the nanoparticles, six 5-FU dry powder formulations containing mixtures of coarse and fine lactose of different percentages were prepared. Deposition of 5-FU was measured using a twin-stage liquid impinger and analyzed using a validated high pressure liquid chromatography method. Addition of fine lactose improved the aerodynamic performance of the drug, as determined by the fine particle fraction.

Ultratrace analysis of uracil and 5-fluorouracil by molecularly imprinted polymer brushes grafted to silylated solid-phase microextraction fiber in combination with complementary molecularly imprinted polymer-based sensor.

Main inborn errors of metabolism diagnosable through uracil (Ura) analysis and the therapeutic monitoring of toxic 5-fluorouracil (5FU) in dihydro pyrimidine dehydrogenase (DPD) deficient patients require a sensitive, reproducible, selective and accurate method. In this work, an artificial receptor in the format of molecularly imprinted polymer (MIP) brush 'grafted to' the surface of sol-gel immobilized on cost-effective homemade solid-phase microextraction (SPME) fibers, individually imprinted with either of Ura and 5FU, was used in combination with a voltammetric sensor duly modified with the same MIP. This combination provided up to 10- and 8.4-fold preconcentrations of Ura and 5FU, respectively, which was more than sufficient for achieving stringent detection limits in the primitive diagnosis of uracil disorders and fluoropyrimidine toxicity in DPD-deficient patients. The proposed method permits the assessment of Ura and 5FU plasma concentrations with detection limits pf 0.0245 and 0.0484 ng mL(-1) (RSD = 1.0-2.5%, S/N = 3), respectively, without any problems of non-specific false-positives and cross-reactivities in complicated matrices of biological samples.

5-Fluorouracil derivatives from the sponge Phakellia fusca.

5-Fluorouracil derivatives were isolated from the marine sponge Phakellia fusca collected around the Yongxing Island of the Xisha Islands in the South Sea of China. Their structures were determined on the basis of spectral analysis and X-ray diffraction.

An HPLC method for the measurement of 5-fluorouracil in human plasma with a low detection limit and a high extraction yield.

High performance liquid chromatographic (HPLC) techniques for the quantification of 5-fluorouracil (5-FU) in human plasma have been reported in the literature, however, a low limit of detection was generally found to result in a comparatively low extraction yield. We have developed a simple, rapid and sensitive HPLC method for the measurement of 5-FU in plasma which provides both a low limit of quantification and a high extraction yield. This method involves the solid phase extraction of 5-FU from a 500 microl plasma sample. The extract is then injected into an HPLC system equipped with a C18 (mu)Bondapak column, and a UV detector set at 260 nm. Ethyl acetate and potassium dihydrogen phosphate are used for the solid phase extraction and the HPLC mobile phase, respectively. This method provides in a good baseline, a sharp and symmetrical peak for 5-FU, and a high resolution between 5-FU and the internal standard. The retention time of 5-FU using this method is 4.7 min with a limit of detection of 5 ng/ml, and an extraction yield of 96.2+/-0.5% (SE). The next injection is possible in 11 min, and the coefficients of variation are 4.2-8.9% for interday precision, and 5.2-10.6% for day-to-day reproducibility. An HPLC method has been developed that has a low limit of detection and a high extraction yield. This technique was successfully applied in a clinical pharmacokinetic study of 5-FU.

Separation of selected anticancer drugs using superheated water as the mobile phase.

Six anticancer drugs have been eluted on a polystyrene-divinylbenzene (PS-DVB) column with buffered superheated water as the mobile phase. The temperature range studied was from ambient temperature up to 160 degrees C, and the pH of the water was adjusted to 11.5 and 3.5 with phosphate buffer. It was possible to separate the substances 5-fluorouracil (5-FU), methotrexate (MTX), 7-hydroxymethotrexate (7-OH-MTX), and etoposide (VP-16) in one chromatographic run. The separation of these substances was optimized when adjusting the pH from 11.5 to 3.5, resulting in a total elution time of less than 13 min. Furthermore, retention factors of all of the investigated substances were measured at different temperatures and pH values.

Hydrophilic interaction chromatography using amino and silica columns for the determination of polar pharmaceuticals and impurities.

Hydrophilic interaction chromatography (HILIC) is described as a useful alternative to reversed-phase chromatography for applications involving polar compounds. In the HILIC mode, an aqueous-organic mobile phase is used with a polar stationary phase to provide normal-phase retention behavior. Silica and amino columns with aqueous-acetonitrile mobile phases offer potential for use in the HILIC mode. An examination of the retention and separation of several pyrimidines, purines, and amides on silica and amino columns from three manufacturers revealed that mobile phases should contain a buffer or acid for pH control to achieve similar and reproducible results among columns from different sources. Amino columns may also be used in an anion-exchange mode, which provides an advantage for some applications. In some cases, silica can provide different selectivity and better separation than an amino column. Example applications include: low-molecular-mass organic acids and amides as impurities in non-polar drug substances, 5-fluorouracil in 5-fluorocytosine, guanine in acyclovir, and different selectivity for polar basic compounds compared to an ion-pairing system.

Estudo comparativo entre o uso do 5-fluoro-uracil e da mitomicina em olhos trabeculectomizados / Comparative study between 5 FU and mitomycin in trabeculctomized eyes

Vinte e oito olhos foram submetidos à trabeculectomia, sendo que em 14 se utilizou o 5-fluoro-uracil (grupo A) e em 14 mitomicina (grupo B). Ambos os grupos apresentavam 6 pseudofácicos, 4 afácicos e 4 fácicos. Os pseudofácicos e afácicos apresentavam uma cirurgia filtrante prévia e os fácicos duas. Todos apresentavam pressöes intra-oculares inaceitáveis, a despeito do uso de terapia máxima tolerável. As pressöes pré-operatórias näo apresentaram diferenças estatisticamente significantes entre o grupo em que se utilizou a mitomicina (12,43 + ou - 6,14) em relaçäo ao grupo em que se utilizou o 5 FU (15,00 + ou - 5,59). A incidência de complicaçöes como atalamia, descolamento de coróide, deiscência de sutura e presença de teste de Seidel positivo foram mais frequentes no grupo em que se utilizou a mitomicina, embora sem diferença estatistica significante. As alteraçöes epiteliais corneanas foram mais frequentes no grupo em que se utilizou o 5 FU. Säo sugeridas algumas mudanças de técnica cirúrgica para se evitar tais complicaçöes

Comparison of the reversed-phase high-performance liquid chromatographic separations of fluoropyrimides, pyrimidines, and purines.

The reversed-phase, high-performance liquid chromatographic separation of fluoropyrimidines, pyrimidines, and purines was investigated under isocratic conditions at ambient temperature. The performance of nine analytical, commercially available columns with five mobile phases is compared, and capacity and resolution factors are reported. The variables determining resolution are discussed, and the systems accomplishing the desired separation of fluoropyrimidine and pyrimidine bases and nucleosides are described. The best chromatographic results are achieved by using Spherisorb ODS-2 as stationary phase and 0.05 M monobasic ammonium phosphate (pH 3.5) as mobile phase.

Reverse-phase liquid chromatography of 5-fluorouracil nucleosides and nucleotides in the presence of quaternary ammonium ions.

Reversed-phase liquid chromatography is used for the separation of 5-fluorouracil, its deoxyribo- and ribonucleosides and nucleotides. The bases and nucleosides are easily separated from their naturally occurring analogues on an octadecyl silica column eluted with 2.10(-2) M KH2PO4 (pH 5.0) containing 5% (v/v) of methanol. This system can be applied to the measurement of 5-fluoro-2'-deoxyuridine serum levels down to 0.1 microgram/ml. Addition of small amounts (10(-3) M) of tetrabutylammonium phosphate to the eluent results in a large retention increase for the nucleotides, while the capacity ratios of the bases and nucleosides remain unchanged. The influence of the tetrabutylammonium phosphate and ammonium phosphate concentrations and pH of the eluent on the various k' values was investigated. Evidence is presented indicating that the quaternary ammonium compound is adsorbed onto the octadecyl silica surface: nucleotides are probably retained as adsorbed tetrabutylammonium ion-pairs.

Application of ion-pair methods of extraction of fluorouracil from aqueous fluids.

The polar molecule, fluorouracil, is a monoanion at pH 10 and may be quantitatively extracted from aqueous solutions with quaternary alkylammonium ions into an organic solvent such as dichloromethane as the ion-pair. Extraction constants of fluorouracil with the tetrapentylammonium ion in dichloromethane or dichloromethane-1-butanol (9:1) and with the tetrahexylammonium ion in dichloromethane were determined. Slope analysis demonstrated that association of the ion components in the aqueous phase occurred as the side reaction. A column ion-pair extraction technique, using tetrapentylammonium as the counterion and dichloromethane as the eluting phase, was developed and allowed quantitative transfer of fluorouracil to the organ solvent. The applicability of this method was shown by determining plasma levels of fluorouracil in cancer patients to whom 1 g of active substance was administered intravenously.