Quantitative analysis of 5-fluorouracil and 5,6-dihydrofluorouracil in plasma by gas chromatography mass spectrometry

5-Fluorouracil and 5,6-dihydro-5-fluorouracil were analysed in the plasma of patients by combined gas chromatography mass spectrometry. 5-Bromouracil was the internal standard. After extraction from plasma with an isopropanol-diethyl ether mixture (20/80) the components were pentylated and the derivatives produced extracted into diethyl ether. Electron impact mass spectrometry was used for the simultaneous quantitative determinations of 5-fluorouracil and 5,6-dihydro-5-fluorouracil (detection limit 10 ng ml-1 5-fluorouracil, 80 ng ml-1 5,6-dihydro-5-fluorouracil). Chemical ionization was utilized to measure 5,6-dihydro-5-fluorouracil concentrations less than 80 ng ml-1 (sensitivity 10 ng ml-1). The biological applicability of these two techniques was demonstrated by analysing plasma samples from patients after administration of 5-fluorouracil or 5'-deoxyfluorouridine by intravenous injections and infusions.     

Determination of methicillin resistance in clinical strains of Staphylococcus aureus in routine microbiological diagnostics

The aim of this study was to compare the results of two methods: disc-diffusion test and ATB STAPH 5 (version 2000) test applied for determination of methicillin-resistant staphylococci. One hundred and twenty S. aureus strains isolated from clinical specimens in microbiological laboratory of Medical University in Warsaw were investigated. Identification and drug resistance were determined using automatic ATB Expression System. We compared the results of methicillin-resistant S. aureus detection by disc-diffusion method (oxacillin 1 microg) and ATB STAPH 5; version 2000 (bioMérieux sa). In the case of 116 strains S. aureus identical results were obtained in both methods, 4 strains gave inconsistent results in these two methods. Results that were obtained show that the ATB STAPH 5 (version 2000) test is as useful for routine determination of MRSA and MSSA strains as the disc-diffusion method, and may be used alternatively.     

ENHANCEMENT OF 5-FLUOROURACIL CYTOTOXICITY BY CISPLATIN IN BRAIN TUMOUR CELL LINES

Cisplatin reportedly plays an important role as a chemical modulator in enhancing the chemotherapeutic effects of 5-fluorouracil on tumour cells. The aim of the present study was to test the synergistic cytotoxicity of cisplatin and 5-fluorouracil in 5-fluorouracil-resistant (C6) and -sensitive (9L) rat brain tumour cell lines. Survival fractions, determined using colony-formation assays, were compared following 5-fluorouracil treatment, with and without cisplatin. The presence of cisplatin (1–10μm ) enhanced cytotoxicity by more than three times compared with 5-fluorouracil alone in 5-fluorouracil-resistant C6 cells, whereas no enhancement effects were noted in 9L cells. These results suggest that a cisplatin-fluorouracil-based regimen may be promising in the treatment of 5-fluorouracil-resistant brain tumours.     

Determination of the concentrations of 5-fluorouracil and its metabolites in rabbit plasma and tissues by high-performance liquid chromatography

The concentrations of 5-fluorouracil, 5-fluoro-5,6-dihydrouracil, 5-fluorouridine and 5-fluoro-2′-deoxyuridine in plasma, liver, kidney, lung and heart of rabbits were determined by high-performance liquid chromatography (HPLC) after drug administration by two different routes. HPLC was carried out by using a Spherisorb 5 ODS 2 column and 0.05 M phosphate buffer as the mobile phase with UV detection at 200 nm. The pH of the mobile phase, organic modifier content and column temperature were found to have a profound influence on the results, hence it was necessary to optimize a procedure for each matrix. A comparison of the efficiency of intravenous and peritoneal administration revealed that the latter provides higher drug concentrations in the liver and minimal contents in plasma and all other tissues studied.     

Manganese(II) complex of 6,7-dicycanodipyridoquinoxaline with antitumor activities: synthesis,crystal structure and binding with DNA

A new Mn(II) complex with the planar ligand 6,7-dicycanodipyrido[2,2-D:2',3'-f]quinoxaline (L) [MnL(NO(3))(H(2)O)(3)]NO(3).CH(3)OH (1) has been synthesized and characterized by elemental analysis, IR, TG-DTA and molar conductance. Its crystal structure was determined by X-ray diffraction, crystal data: yellow, triclinic, space group P1;, Z=2, a=7.3743(8) A, b=11.2487(15) A, c=14.1655(15) A, alpha=79.412(2) degrees, beta=83.208(2) degrees, gamma=80.466(2) degrees. The Mn atom was hexa-coordinated to form a distorted octahedral geometry by two nitrogen atoms of L and four oxygen atoms of three H(2)O and NO(3)(-) in the complex. The binding mode of the complex with calf thymus DNA has also been investigated with spectrophotometric methods, viscosity and thermal denaturation measurements. The experimental results indicate that the complex intercalated into DNA base pairs via the ligand L. The intrinsic binding constant K(b) values for 1 (5.00 x 10(5) M(-1)) and L (1.65 x 10(5) M(-1)) were determined by absorption titration and calculated with the model of McGhee and Von Hippel. Biological tests against four different cell lines (HL-60, KB, Hela and BGC-823) in vitro showed that the complex had significant antitumor properties since the 50% inhibition concentrations (IC(50)) of the complex were within a microM range similar to those of antitumor drug 5-fluorouracil.     

Gas-liquid chromatographic analysis of fluoropyrimidine nucleosides and fluorouracil in plasma and urine

A gas-liquid chromatographic method employing on-column alkylation and a nitrogen-sensitive detector was developed for the analysis of 5-fluoro-2'-deoxyuridine, 5-fluorouridine, and 5-fluorouracil in plasma and urine. Samples (0.72 ml) containing the fluoropyrimidine and internal standard (5-chloro-2'-deoxyuridine for nucleoside analyses and 6-methyluracil for 5-fluorouracil analyses) were prepared for gas-liquid chromatography by sequential cation-exchange and anion-exchange column chromatography. Recoveries of fluoropyrimidines were 71-95% over the concentration ranges studied. The dried eluate from the anion-exchange column was dissolved in p-tolyltrimethylammonium hydroxide in methanol before gas-liquid chromatographic analysis. Columns packed with either 3% SP-2100 on Supelcoport or 3% OV-1 on Gas-Chrom Q were suitable for nucleoside analyses; a column packed with 0.75% Carbowax 20M-5% KOH on Chromsorb G was used for 5-fluorouracil analyses. The fluoropyrimidine nucleosides were well separated from each other and from the potentially interfering endogenous compounds 2'-deoxyuridine and uridine; 5-fluorouracil was well separated from uracil. Linear standard curves (peak area ratio method) were obtained for plasma containing 0.025 to 20 micrograms FdUrd (0.1 to 81 microM) or 0.05 to 1.0 microgram FUrd (0.2 to 3.8 microM), and for urine containing 0.2 to 1.0 microgram (0.8 to about 4 microM) of the nucleosides. Standard curves for 5-fluorouracil (1.5 to 7.9 microM) and 2'-deoxyuridine (0.9 to 4.4 microM) were also linear. A measurable amount of 5-fluorouracil, equivalent to 4 to 7% of the 5-fluoro-2'-deoxyuridine injected, was formed from the nucleoside on the gas-liquid chromatographic column, requiring correction of 5-fluorouracil concentrations measured in the presence of 5-fluoro-2'-deoxyuridine.     

Genetic and Physiological Aspects of Resistance to 5-Fluoropyrimidines in Saccharomyces cerevisiae

Mutants resistant to 5-fluorouracil, 5-fluorocytosine, and 5-fluorouridine were selected in yeast, and the mechanisms of their resistance were investigated. The investigated mutations map in seven different loci. (i) A mutation at the locus FUI 1 gives specifically resistance to 5-fluorouridine. (ii) Two loci are involved in a specific 5-fluorocytosine resistance: a mutation at locus FCY 1 produces a loss of cytosine deaminase activity; a mutation at locus FCY 2 results in the loss of the activity of a cytosine-specific permease. (iii) A mutation at the locus FUR 4 gives a simultaneous resistance to 5-fluorouracil and to 5-fluorouridine by loss in the activity of the uracil-specific permease. (iv) We found three types of mutants in the locus FUR 1. One is dominant and weakly resistant to 5-fluorouracil, 5-fluorocytosine, and 5-fluorouridine. The two others are recessive and are unable to catalyze one of the steps involved in uracil transformation into uridine 5'-monophosphate; this block-age explains their strong resistance to 5-fluorouracil and 5-fluorocytosine. Of these two mutants, one is resistant to 5-fluorouridine and the other is not. (v) Mutations at locus FUR 2 give resistance to 5-fluorouracil, 5-fluorocytosine, and 5-fluorouridine. These mutations are dominant and lead to a loss in the feedback regulation of the aspartic transcarbamylase activity by uridine triphosphate. (vi) The mutants FUR 3 are resistant to 5-fluorocytosine and 5-fluorouridine. They are dominant and physiologically related to the mutants of the locus FUR 1 but their mechanism of resistance is not understood.     

Topical Delivery of 5-Fluorouracil from Pheroid™ Formulations and the In Vitro Efficacy Against Human Melanoma

Drug delivery vehicles can influence the topical delivery and the efficacy of an active pharmaceutical ingredient (API). In this study, the influence of Pheroid™ technology, which is a unique colloidal drug delivery system, on the skin permeation and antimelanoma efficacy of 5-fluorouracil were investigated. Lotions containing Pheroid™ with different concentrations of 5-fluorouracil were formulated then used in Franz cell skin diffusion studies and tape stripping. The in vitro efficacy of 5-fluorouracil against human melanoma cells (A375) was investigated using a flow cytometric apoptosis assay. Statistically significant concentrations of 5-fluorouracil diffused into and through the skin with Pheroid™ formulations resulting in an enhanced in vitro skin permeation from the 4.0% 5-fluorouracil lotion (p < 0.05). The stratum corneum-epidermis and epidermis-dermis retained 5-fluorouracil concentrations of 2.31 and 6.69 μg/ml, respectively, after a diffusion study with the 4.0% Pheroid™ lotion. Subsequent to the apoptosis assay, significant differences were observed between the effect of 13.33 μg/ml 5-fluorouracil in Pheroid™ lotion and the effects of the controls. The results obtained suggest that the Pheroid™ drug delivery system possibly enhances the flux and delivery of 5-fluorouracil into the skin. Therefore, using Pheroid™ could possibly be advantageous with respect to topical delivery of 5-fluorouracil.KEY WORDS: A375 cells, cell culture, flow cytometry, melanoma, permeation enhancer     

Synthesis of FUDP-N-acetylglucosamine and FUDP-glucose in Saccharomyces cerevisiae cells treated with 5-fluorouracil

Saccharomyces cerevisiae cells (strain W303-1A) treated with 5-fluorouracil and grown in 2% (fermentative conditions) or in 0.1% glucose (oxidative conditions) accumulated two types of 5-fluoro-UDP-sugars (FUDP-sugars): FUDP-N-acetylglucosamine and FUDP-glucose. No difference was observed in both conditions of culture. The viability of yeast cells on treatment with 5-fluorouracil was also followed. Both FUDP-sugars were partially purified by column chromatography (on Hypersil ODS and Mono Q columns) and characterized by: (i) treatment with alkaline phosphatase (EC 3.1.3.1), snake venom phosphodiesterase (EC 3.1.4.1) and UDP-glucose dehydrogenase (EC 1.1.1.22); (ii) UV spectra; and (iii) matrix-assisted laser desorption/ionization-time of flight mass analysis and 1H-nuclear magnetic resonance spectrometry. The syntheses of both FUDP-sugars were inversely related to the concentration of uracil and directly related to the concentration of 5-fluorouracil in the culture medium. The strain W303-1A, requiring uracil for growth, was useful as a tool to analyze the effect of 5-fluorouracil on nucleotide metabolism.     

Characterisation of a partially purified uracil phosphoribosyltransferase from the opportunistic pathogenCandida albicans

This paper describes for the first time the partial purification and properties of uracil phosphoribosyltransferase (UPRTase) from the yeastCandida albicans. UPRTase was purified 38 fold by acid precipitation, DEAE-Sephacel chromatography and ultrafiltration. Further purification of UPRTase was unsuccessful due to the labile nature of the enzyme and the failure in obtaining satisfactory stabilizing conditions. SDS-PAGE suggested that the enzyme exists as a dimer of two dissimilar subunits with molecular masses of 47 and 38 kDa. The pH optimum for phosphoribosylation was about 7.5 and the optimal Mg⁺⁺ concentration was 2 mM. The kinetics of the enzymes for its substrates, uracil and 5-phosphoribosyl-1-pyrophosphate (PRPP) were determined by measuring initial enzyme velocities over a wide range of concentrations of either substrate at different fixed concentrations of the second substrate. Graphic analysis of the data by Hanes-Woolf plots indicated that the reaction is indistinguishable from a double displacement reaction. Ping pong mechanism has been previously reported for other phosphoribosyltransferases. The enzyme has a low affinity for its substrates (K _m=70.5 and 186 µM for uracil and PRPP, respectively) as compared with those ofE. coli and baker's yeast. Inhibition studies indicate that 5-fluorouracil acts as an alternative substrate for UPRTase with 1.6 times higher specific activity.Abbreviations UPRTase Uracil phosphoribosyltranferase - PRTases phosphoribosyltransferases - PRPP 5-phosphoribosyl-1-pyrophosphate - 5-FC 5-fluorocytosine - 5-FU 5-fluorouracil - PEI polyethyleneimine - DTT dithiothreitol - DMSO dimethyl sulphoxide - PMSF phenylmethylsulphonyl fluoride - UMP uridine mono-phosphate     

Pharmacokinetics of dioxidine in oncological patients in the postoperative period

Dioxidine pharmacokinetics was studied in 5 patients operated for cancer of the large intestine and treated prophylactically with the drug during the postoperative period. Dioxidine was administered intravenously for 10 minutes twice a day in an amount of 300 mg in 5 per cent solution of glucose. The drug concentrations in serum and urine were determined with a microbiological procedure. Escherichia coli AB 2472 rec A16, a strain deficient with respect to reparation was used as a test microbe. The plates with the dilutions were incubated under anaerobic conditions. The time course of the drug concentrations in serum was shown to be satisfactorily described by the following equation: C(t) = 3.125 . 1-2.57.t + 2.76 . 1-0.64.t. Within the first 1.5-2 hours after the administration the dioxidine concentrations in serum and urine amounted to 2.5-4 and 35-50 micrograms/ml respectively.     

Synthesis and properties of lipophilic derivatives of 5-fluorouracil

A series of N ¹-acyl derivatives of 5-fluorouracil (5-FU) bearing the residues of palmitic, p-myristoylaminobenzoic, p-oleoylaminobenzoic, and adamantane-1-carboxylic acids have been synthesized. The relative hydrolysis rates for the derivatives under physiological conditions (pH 7.2 and 37°C) have been determined, and it has been shown that the resistance of these compounds to hydrolysis increases as the steric accessibility of the amide group at residue N ¹ of 5-FU decreases. The derivatives easily incorporate into the lipid bilayer; their liposomal preparations show a marked cytostatic activity on human breast lymphoma cells (LD50 ～1 μM) and are of interest as potential antitumor preparations. In addition, a fluorescent analogue of the above derivatives, 1-[8-(3-perylenyl)octanoyl]-5-fluorouracil, has been synthesized, which is intended for studying the behavior of 5-FU derivatives in cells and tissues by instrumental methods.     

Isolation and characterization of cultured mouse T-lymphoma cells deficient in uridine-cytidine kinase.

Two clones were isolated from mutagenized mouse T-lymphoma cells (S49) which are over 90% deficient in uridine-cytidine kinase. The first clone, AU-200-1, was isolated in two steps by virtue of its resistance to 6-azauridine; whereas the second clone, FU3-70G, was isolated in three steps after exposure to three increasing concentrations of 5-fluorouracil. Extracts of both the AU-200-1 and the FU3-70G cell lines lacked over 90% of the capacity of those from wild type cells to phosphorylate either uridine or cytidine. Furthermore, the uptake of radioactive uridine and cytidine from the medium by intact AU-200-1 and FU3-70G cells was less than 5% of that found for intact wild type cells. By growth rate experiments, these uridine-cytidine kinase-deficient cell lines have altered sensitivities to the toxic pyrimidine analogs, 6-azauridine, 5-fluorouracil, and 5-fluorouridine and thus have been useful in elucidating the biochemical determinants involved in the metabolism of these compounds.     

Comparative Study of Responses to Neomycins B and C by Microbiological and Gas-Liquid Chromatographic Assay Methods

The relative responses of neomycins B and C have been determined by a microbiological agar-diffusion method, a turbidimetric method, and by a recently developed gas-liquid-chromatographic (GLC) method capable of separating the neomycin isomers. The ratios of response of neomycin C to neomycin B by the individual methods were as follows: agar-diffusion method, 1:3; turbidimetric method, 1:2.5; and GLC method, 1:1. When neomycin C is assumed to have 35% biological activity of neomycin B, the calculated drug contents of neomycin sulfate powders obtained by the GLC method correlated well with values obtained by the microbiological agar-diffusion assay method.     

Rapid determination of 5-fluorouracil in plasma using capillary electrophoresis

A rapid, simple and sensitive capillary electrophoresis (CE) method used for the determination of 5-fluorouracil in rabbit plasma is described in the present paper. In this method, samples were simply pretreated by a solvent extraction procedure prior to injection. With a running buffer composed of 30 mM Tris-H(3)PO(4) (pH 7.0) and 5% isopropanol, 5-fluorouracil was easily separated from the external standard alpha-phenethylol as well as other substances existed in the plasma. A linearity of 5-fluorouracil was determined in the range from 0.17 to 42.50 microg/ml with a correlation coefficient of 0.999. A limit of quantitation (LOQ) corresponding to signal-to-noise ratio of 10 was obtained (LOQ=0.08 microg/ml). The method was successfully used for determining the 5-fluorouracil in real plasma samples from rabbits.     

High-performance liquid chromatographic assay of 5-fluorouracil in human erythrocytes,plasma and whole blood

A HPLC assay method was modified and validated for the determination of 5-fluorouracil in human red blood cells, plasma and whole blood with a two-fold increased sensitivity (detection limit=10 ng/ml). The assay was linear from 25 to 1500 ng/ml and the accuracy ranged from 96.7 to 103.2% at 25 ng/ml, 94.8 to 99.4% at 500 ng/ml, and 98.9 to 99.5% at 1500 ng/ml. Intra-assay and inter-assay coefficients of variation were less than 8% over the range of concentrations and less than 8% over 10 days of analysis. After intravenous bolus and infusion of 5-fluorouracil in patients with colorectal cancer, the concentrations of 5-fluorouracil in whole blood were 108–111% of plasma concentrations, while packed red blood cells levels were 8–15% of plasma concentrations in the five patients studied. By utilising basic analytical hardware, this represents an accurate, precise, reproducible and affordable method for 5-fluorouracil pharmacokinetics investigation and therapeutic drug monitoring.     

Inhibition of Light-Dependent Development of Chloroplasts by 5-Fluorouracil

The uracil analogue, 5-fluorouracil, inhibited the developmentof chloroplasts in Euglena gracilis, strain Z. Chlorophyll synthesiswas inhibited when dark-grown cells were illuminated in thepresence of 5-fluorouracil, but only if the 5-fluorouracil waspresent during the lag phase of chlorophyll synthesis. Ribonucleaseshowed a similar inhibition. Equimolar concentrations of uracilreleased inhibition by 5-fluorouracil, but if cells were incubatedin the light with 5-fluorouracil before addition of uracil,the ability of uracil to effect rapid reversal of 5-fluorouracilinhibition was decreased. In contrast, prior incubation with5-fluorouracil in the dark did not affect reversibility by uracil.The synthesis of a chloroplast-localized protein, cytochromec (552, Euglena), was also inhibited by 5-fluorouracil, whereasthe light-stimulated synthesis of a number of cytoplasmic enzymeswas enhanced. The results suggest that addition of 5-fluorouracilat the beginning of the illumination period preferentially interfereswith the synthesis of chloroplast protein compared with thesynthesis of cytoplasmic protein by inhibiting the formationof a ribosomal system, presumably localized in the chloroplast,that functions in the synthesis of chloroplast protein. Thedata also suggest that in uninhibited cells, the formation ofthis ribosomal system was largely completed within the first10 to 14 h of illumination and before the main period of synthesisof chloroplastproteins.     

Isolation and Partial Characterization of Regulatory Mutants of the Pyrimidine Pathway in Salmonella typhimurium

Mutants of Salmonella typhimurium affected in the regulation of pyrimidine biosynthesis were isolated by two methods. The first involved screening for bacteria able to feed a pyrimidine-requiring indicator strain, and the second involved selection for bacteria simultaneously resistant to two pyrimidine analogues, 5-fluorouracil and 5-fluorouridine, in a S. typhimurium strain unable to degrade 5-fluorouridine. Among the mutants isolated by these methods are constitutive mutants, producing high levels of pyrimidine biosynthetic enzymes in the presence or absence of pyrimidines, and feedback modified mutants, in which aspartate transcarbamylase is partially desensitized to its inhibitor, cytidine triphosphate. No fully desensitized mutant has been found. The partially desensitized character cotransduces with the pyrB locus, that of aspartate transcarbamylase. The constitutive character has been determined in a few cases to be localized in the region of leu and pro on the Salmonella map.     

A study of trace and minor elements in the solid tumor model 3924A hepatoma before and after treatment with 5-fluorouracil

The changes in the concentrations of a number of trace elements have been determined by neutron activation analysis in a solid tumor model, blood and its host liver, following 5-fluorouracil administration. Studies have also been carried out for non-tumor bearing animals after 5-fluorouracil. The changes in some of the trace elements parallel destructive changes found in the tumor. Studies are in progress to determine if changes in the urinary concentrations of these trace elements parallel the time sequence of the destructive changes found in the tumor. The potential clinical usefulness of these trace elements are being evaluated along with other biological markers which may eventually be utilized to assess the therapeutic effectiveness of different treatment modalities as well as monitor the patient for possible reoccurrence of the cancer following treatment.     

Effects of some drugs on purified human erythrocyte CuZnSOD and in vitro inhibitiory effect of 5-fluorouracil on leukocyte total SOD activity

The inhibition and activation effects of some drugs on the activities of superoxide dismutase enzymes (SOD) in human erythrocyte and leukocyte cells was investigated. Firstly, CuZnSOD enzyme was purified 837–fold and 12% efficiency from human erythrocytes by ethanol-chloroform treatment to remove hemoglobin and then ion exchange chromatography (DEAE-Sepharose) and copper chelate affinity chromatography techniques. Inhibition or activation effects of fourteen drugs on CuZnSOD was investigated. None of the studied drugs except for 5-fluorouracil showed any effects on the enzyme. 5-fluorouracil showed activation effects on CuZnSOD at 3.33 mg/ml and 4 mg/ml concentrations with 33% and 32% activation, respectively. Leukocytes were isolated from healthy human blood, lysed in liquid nitrogen and the effect of 5-fluorouracil on the lysate SOD activity investigated. 5-Fluorouracil showed inhibition effects on total SOD activity of human leukocytes at 2 mg/ml and 4 mg/ml concentrations with 42% and 62% inhibition, respectively.