Concentrations Of 5-fluorouracil Research Articles

Pharmacokinetic application of a bio‐analytical LC‐MS method developed for 5‐fluorouracil and methotrexate in mouse plasma, brain and urine

In the past we have reported significant cognitive deficits in mice receiving 5-fluorouracil in combination with low-dose methotrexate. To explain such interactions, a pharmacokinetic study was designed. A sensitive bio-analytical method was therefore developed and validated for 5-fluorouracil and methotrexate in mouse plasma, brain and urine with liquid chromatography coupled to a single quadrupole mass spectrometer. Chromatographic separation was accomplished by Agilent® Zorbax® SB-C18 column, with isocratic elution (5 mM ammonium acetate and methanol, 70:30, %v/v) at a flow rate of 300 μL/min. The limit of quantitation for both drugs was 15.6 ng/mL (plasma and brain) and 78.1 ng/mL (urine), with interday and intraday precision and accuracy ≤15% and a total run time of 6 min. This bio-analytical method was used for the pharmacokinetic characterization of 5-fluorouracil and methotrexate in mouse plasma, brain and urine over a period of 24 h. This method allowed characterization of the brain concentrations of 5-fluorouracil over a period of 24 h.

Cytostatic and Cytotoxic Effects of 5-Fluorouracil on Human Corneal Epithelial Cells and Keratocytes

To investigate the effects of various 5-fluorouracil (5-FU) concentrations, exposure times, and application techniques on in vitro-cultured human corneal cells. Human corneal epithelial cell (HCEC) and human corneal keratocyte (HCK) cultures were exposed to different 5-FU concentrations (0.025%-1%) and incubation durations (5 minutes to 2 hours). The cytostatic effect was evaluated as the percentage of inhibition of migration relative to the control. The evaluation of cytotoxic effect included both phase contrast microscopic observations and viability measures performed using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] colorimetric assay. The results are expressed as ratio of optical density (OD) reduction 24 hours after exposure. The cytostatic effect was time and dose dependent. The 50% inhibiting dose was 0.55% after 1 hour of incubation for HCECs and was 0.5% after 2 hours of incubation for HCKs. A 100% inhibitory effect was never observed at any concentration or incubation duration. No cytotoxic changes were observed using an 5-FU concentration of <1%; 1% 5-FU showed time-dependent cytotoxic changes in HCEC cultures only. MTT analysis showed no OD reduction at 5-FU concentrations of <1%, whereas 1% 5-FU showed OD reduction <50% at any tested exposure time. HCECs showed higher reduction in OD than HCKs. 5-FU formulations topically used in clinical practice showed limited toxicity in normal cultured corneal epithelial cells and keratocytes.

Paraptosis and NF-κB activation are associated with protopanaxadiol-induced cancer chemoprevention

BackgroundProtopanaxadiol (PPD) is a triterpenoid that can be prepared from steamed ginseng. PPD possesses anticancer potential via caspase-dependent apoptosis. Whether paraptosis, a type of the caspase-independent cell death, is also induced by PPD has not been evaluated.MethodsCell death, the cell cycle and intracellular reactive oxygen species (ROS) were analyzed by flow cytometry after staining with annexin V/PI, PI/RNase or H2DCFDA. We observed morphological changes by crystal violet staining assay. Mitochondrial swelling was measured by ultraviolet–visible spectrophotometry. The activation of NF-κB was measured by luciferase reporter assay.ResultsAt comparable concentrations of 5-fluorouracil, PPD induced more cell death in human colorectal cancer cell lines HCT-116 and SW-480. We demonstrated that PPD induced paraptosis in these cancer cells. PPD treatment significantly increased the percentage of cancer cells with cytoplasmic vacuoles. After the cells were treated with PPD and cycloheximides, cytoplasmic vacuole generation was inhibited. The paraptotic induction effect of PPD was also supported by the results of the mitochondrial swelling assay. PPD induced ROS production in cancer cells, which activated the NF-κB pathway. Blockage of ROS by NAC or PS-1145 inhibited the activation of NF-κB signaling.ConclusionsPPD induces colorectal cancer cell death in part by induction of paraptosis. The anticancer activity of PPD may be enhanced by antioxidants such as green tea, which also inhibit the activation of NF-κB signaling.

Bioequivalence Studies of A Generic Formulation (SW651K) to the Brand Drug S-1 in Tumor-Bearing Rat Models

SW651K, a fixed combination of tegafur (FT), 5-chloro-2,4-dihydroxypyridine (CDHP) and potassium oxonate (Oxo), is a generic preparation of S-1, which is widely used to treat gastric cancer in Japan. There is little detailed information of pharmacological effects of SW651K in clinical therapy. However it is not easy to evaluate the PK/PD in clinical trial. Therefore this study examined the bioequivalence of SW651K to S-1 in terms of pharmacokinetics and tumor shrinkage in tumor-bearing rats. Bioequivalence of SW651K to S-1 was first evaluated in Yoshida sarcomabearing rats. Concentrations of FT, 5-fluorouracil (5-FU, the active metabolite of FT), CDHP, and Oxo in plasma, tumor, small intestine, and large intestine were analyzed after a single dose of SW651K. Tumor size was measured during treatment with each formulation for 7 consecutive days. Next, tumor size was measured in human gastric cancer cell (NUGC4)-bearing rats treated for 14 days. Tumor 5-FU concentrations were also analyzed. Tumor size in NUGC4-bearing rats treated with SW651K or S-1 plus cisplatin was also evaluated. SW651K was bioequivalent to S-1 in terms of the pharmacokinetics of all components and of 5-FU in rats. Both formulations also had equivalent antitumor activities in Yoshida sarcoma- and NUGC4 tumor-bearing rats that received monotherapy. Moreover, combined treatment with cisplatin equally potentiated the antitumor effects of both formulations, without increasing body-weight loss in NUGC4 tumor-bearing rats. In conclusion, bioequivalence of SW651K and S-1 was confirmed in terms of pharmacokinetics and antitumor effectiveness in tumor-bearing rats. Our results suggest that SW651K is clinically equivalent to S-1.

155P - Effect of Bevacizumab on Tumour 5-Fluorouracil Concentration and Microcirculatory Parameters Obtained by Dynamic Contrast-Enhanced MRI in a Hepatocellular Carcinoma Xenograft Model

ABSTRACT Introduction Vascular endothelial growth factor (VEGF) is expressed by tumours to promote angiogenesis. Clinical trials of anti-VEGF therapy combined with chemotherapy demonstrate improvement in survival. We investigate the effect of bevacizumab (bev), a humanized monoclonal antibody that inhibits VEGF-A, on tumour 5-fluorouracil (5FU) concentration in a hepatocellular carcinoma (HCC) xenograft model, together with changes in tumour microcirculatory parameters obtained by dynamic contrast-enhanced (DCE) MRI. Methods 48 immunodeficient mice implanted with subcutaneous HCC xenografts were first injected with bev (1mg/kg or 10mg/kg) or omalizumab (control). 1 day later, they received 5FU and were sacrificed. Tumour and plasma 5FU concentration was measured by liquid chromatography-mass spectrometry. A subset of 17 mice underwent DCE MRI with gadolinium contrast at baseline and one day after bev/omalizumab injection. Scans employed a three-dimensional spoiled gradient recalled sequence with tracer concentration estimated using variable flip-angle technique. Data analysis employed a conventional two-compartment tracer kinetic model. Results Tumour 5FU concentration (µg/g) was significantly lower 1 day after bev (10mg/kg) compared to control (8.4 vs 21.6, p = 0.027). However, there was no significant difference in plasma 5FU concentration between the bev and control groups. Following bev (10mg/kg), DCE MRI measurements of tumour intravascular blood volume reduced by 38.3% (p = 0.01); there was no significant change in DCE-MRI parameters in the control group. Following bev (1mg/kg), tumour permeability-surface area product (ml/100ml/min) correlated with tumour 5FU concentration (r2 = 0.84, p = 0.01). Conclusion Bev causes vascular shutdown consistent with its anti-angiogenic mode of action. Reduced tumour 5FU concentration suggests that the reported synergism between anti-VEGF and chemotherapy may not be attributable to improved drug delivery at this early time point. DCE-MRI may play a role as a biomarker for tumour drug concentration following anti-VEGF therapy. Disclosure All authors have declared no conflicts of interest.

Effect of gene GSTP1 silencing via shRNA transfection on androgen independent prostate cancer cell line Du145

To design short hairpin RNA (shRNA) interference sequence to silence glutathione S-transferase P1 (GSTP1) gene of androgen independent prostate cancer cell line DU145, and to explore its effect on proliferation and sensitivity to chemotherapeutics. The target sequence was picked up to form the shRNA, and the 3 shRNA expression vectors were shRNA255, shRNA554 and shRNA593. The DNA template was cloned to plasmid pGPU6/GFP/Neo. The shRNA was identified by enzyme digesting and gene sequencing. The screening experiment was done to pick up the shRNA expression vector with the highest transfection ratio and best gene silencing results. DU145 cells were divided into a blank plasmid group and a shRNA transfected group. According to the chemotherapeutics the DU145 cells were divided into a fluorouracil (FU) group and a paclitaxel (PA) group, and the 2 groups were subdivided into 4 subsets according to the chemotherapeutic concentrations (FU: 30, 60, 120, and 240 μg/mL; PA: 0.2, 2, 10, and 20 μg/mL), meanwhile a blank control group was included respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate the proliferation after the transfection. MTT and terminal de-oxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were used to detect the inhibition effect of different concentrations of 5-FU or PA on the proliferation and induction of apoptosis of DU145. The transfection ratio of the 3 shRNA expression vectors (shRNA255, shRNA554, and shRNA593) was (63.30±1.04)%, (76.20±0.68)%, and (72.70±0.33)%, and the transfection ratio of shRNA554 was the highest. there was significant difference among the above 3 shRNA expression vectors (P<0.01). After the transfection, the mRNA was 128.31±2.50, 43.24±4.30 and 85.62±6.30, the GSTP1 protein was 163.92±12.40, 65.38±9.30 and 114.25±16.70. After the transfection of shRNA554, the mRNA and protein of GSTP1 were the lowest level. there was significant difference among the above 3 shRNA expression vector (P<0.01). MTT analysis showed that before the transfection, the survival ratio of cells under different concentrations of FU (30, 60, 120, and 240 μg/mL) was (95.60±2.11)%, (90.20±0.86)%, (83.10±3.12)% and (74.60±1.32)%; however after the transfection, the survival ratio of cells was (91.30±1.43)%, (84.60±2.13)%, (73.20±1.52)%, and (65.5±0.942)%. TUNEL assay showed that before the transfection, the apoptosis ratio of cells under different concentrations of FU (30, 60, 120, and 240 μg/mL) was (5.50±0.88)%, (10.20±1.64)%, (15.20±2.39)%, and (25.10±2.59)%; however after the transfection, the apoptosis ratio of cells was (10.8±0.62)%, (15.7±1.32)%, (20.4±1.89)%, and (34.9±2.54)%. After the transfection, the cell survival ratio decreased under the same concentration of FU, and the apoptosis ratio increased, with statistical significance (both P<0.01). MTT analysis showed that before the transfection, the survival ratio of cells under different concentrations of PA (0.2, 2, 10, and 20 μg/mL) was (98.50±2.34)%, (95.20±1.32)%, (89.40±0.68)%, and (82.70±1.73)%; after the transfection the survival ratio of cells was (94.20±0.78)%, (86.50±2.13)%, (78.70±1.34)%, and (70.10±0.76)%. TUNEL assay showed that before the transfection, the apoptosis ratio of cells under different concentrations of PA (0.2, 2, 10, and 20 μg/mL) were (2.40±1.07)%, (5.20±1.33)%, (10.50±2.41)%, (20.70±1.92)%; after the transfection the apoptosis ratio of cells was (5.46±2.13)%, (13.80±1.24)%, (21.20±2.39)%, and (29.20±2.21)%. After the transfection, the cell survival ratio decreased under the same PA concentration, and the apoptosis ratio increased, with statistical significance (both P<0.01). gene GSTP1 silence via shRNA transfection to androgen independent prostate cancer cell line DU145 can inhibit its proliferation in time dependent manner, and induce apoptosis and raise its sensitivity to chemotherapeutics.

Long-term three-dimensional cell culture and anticancer drug activity evaluation in a microfluidic chip

In this work, we present a microfluidic array of microwells for long-term tumor spheroid cultivation and anticancer drug activity evaluation. The three-dimensional microfluidic system was obtained by double casting of poly(dimethylsiloxane). Spheroids of HT-29 human carcinoma cells were cultured in the microsystem for four weeks. After two weeks of the culture growth slowdown and stop were observed and high cell viability was determined within next two weeks. The characteristics of a homeostasis-like state were achieved. A cytostatic drug (5-fluorouracil) was introduced into the microsystem with different frequency (every day or every second day) and different concentrations. The geometry and construction of the microsystem enables flushing away of unaggregated (including dead) cells while viable spheroids remain inside microwells and decreasing spheroid diameter can be observed and measured as an indicator of decreasing cell viability. The results have shown differences in response of spheroids to different concentrations of 5-fluorouracil. It was also observed, that higher frequency of drug dosing resulted in more rapid spheroid diameter decrease. The presented microfluidic system is a solution for cell-based studies in an in vivo-like microfluidic environment. Moreover, observation of decreasing spheroid dimensions is a low-cost, label-free and easy-to-conduct mean of a quantitative determination of a 3D cellular model response to a applied drug. It is suitable for long-term observation of spheroid response, in a contrary to other viability assays requiring termination of a culture.

Synergy effects of octreotide combined with cisplatin and 5-fluorouracil on inhibition of lung adenocarcinoma cell line A549

Objective To investigate whether octreotide,as somatostatin analogue,can enhance the sensitivity of the human lung adenocarcinoma cell line A549 to chemotherapeutic drugs.Methods Different concentration of octretide,cisplatin and 5-Fluorouracil (5-Fu) was respectively acted on the lung adenocarcinoma cell line A549.The absorbance value was tested by colorimetry through MTT method to evaluate the effect of octreotide,cisplatin,5-Fu or the three drugs combined respectively after 48 hours.Each drug concentration had six holes and it repeated three times.The effects of combination therapy was analysed with isobologram.Results It was proved that octreotide could inhibit the proliferation of A549 cells in a dose-dependent manner at the concentration range of 1.3 mg/L ～ 166.7 mg/L.The inhibition rate was dose-dependent which was higher when octreotide combined with cisplatin and 5-Fu than it alone.It has statistically significant difference (P ＜ 0.05 ).The effect plots of IC50 were located in the synergy areas of isobologram.Conclusion It can be concluded that octreotide could inhibit the proliferation of A549 cells in vitro.This inhibition enhances when octreotide is combined with cisplatin and 5-Fu.Octreotide can enhance the susceptibility of A549 cells to cisplatin and 5-Fu. Key words: Carcinoma, non-small cell lung; Octreotide; Drug synergy; Isobologram

Efficiency of colon cancer RNA-transfected DC vaccine is enhanced by previous treatment of tumor cells with low concentration of 5-fluorouracil (162.11)

Abstract We have recently observed that human DCs treated with low doses of antitumor chemotherapeutic agents became them more efficient to stimulate T lymphocytes, whereas treatment of tumor cells with low concentrations of drugs made them more immunogenic than wild type cells. In this study we aimed to evaluate whether transfection of DC with drug-treated tumor cells RNA, enhances the effectiveness of DC-based vaccine, and if modulatory effects of drugs can be observed in vivo. To achieve these goals, C57/Bl-6 mice were s.c inoculated with MC-38 cells and 10 days later they were treated with DC transfected with RNA from MC-38 cells pre-treated with the minimum effective concentration of 5-fluorouracil (5-FU). Vaccination with these DC decreased the tumor growth, being the tumor size 40% lower than the Control. DC phenotyping showed that transfection increased the percentage of CD86+ (55% higher than control), CD40+ (57% higher), and MHC class II+ cells (58% higher). Spleen cells of tumor-bearing mice were co-cultured with MC-38 target cells and the supernatants evaluated for IFN-γ. Vaccination with transfected-DC was able to increase the in vitro production of IFN, compared with control. Our results suggest that treatment of tumor cells with minimum effective concentration of 5-FU induces transcriptional changes that can be transfered to DC by RNA transfection, enhancing their ability to stimulate the antitumor response.

Influence of resistance to 5-fluorouracil and tomudex on [18F]‑FDG incorporation, glucose transport and hexokinase activity

Drug resistance is a major obstacle to cancer cure and may influence [18F]-fluorodeoxyglucose (FDG) incorporation. In this study, glucose transport, hexokinase activity and [18F]-FDG incorporation were measured in drug-resistant tumour cells generated by exposing H630 colon and MCF7 breast cancer cells to increasing concentrations of tomudex (raltitrexed) or 5-fluorouracil (5FU). Drug sensitivity was determined using the XTT assay: Tomudex-resistant (H630TDX and MCF7TDX) cells were more than 40,000-fold less sensitive to tomudex than were the parental wild-type, H630WT and MCF7WT cells, respectively. 5FU-resistant (H630R10) cells were 100-fold less sensitive than parental H630WT cells to 5FU. As previously reported for 5FU-resistant MCF7 breast cancer cells, [18F]-FDG incorporation was decreased in H630R10 colon cancer cells compared to the parental line. By contrast, both tomudex-resistant cell lines exhibited increased [18F]-FDG incorporation compared with the parental lines. H630R10 and MCF7TDX cells exhibited higher rates of glucose transport, measured as the initial rate of O-methyl-glucose (OMG) uptake, compared to wild-type cells; however, glucose transport was not significantly different between H630TDX cells and the parental cells. Hexokinase activity was lower in H630R10 and MCF7TDX cells compared with sensitive parental cells but unchanged in H630TDX cells. In conclusion, our results show that [18F]-FDG incorporation is influenced by resistance to antifolate and fluoropyrimidine-based anti-cancer drugs in a drug-dependent manner and the underlying mechanisms appear to be cell- and drug-dependent. Glucose transport may be a useful marker of resistance to 5FU.

An Investigation of Oxidative DNA Damage in Pharmacy Technicians Exposed to Antineoplastic Drugs in Two Chinese Hospitals Using The Urinary 8-OHdG Assay

ObjectiveTo investigate oxidative DNA damage in pharmacy technicians preparing antineoplastic drugs at the PIVAS (Pharmacy Intravenous Admixture Service) in two Chinese hospitals. MethodsUrinary 8-OHdG served as a biomarker. 5-Fluorouracil (5-FU) concentrations in air, masks and gloves were determined. The spill exposure of each PIVAS technician to antineoplastic drugs was investigated. Eighty subjects were divided into exposed group I, II, and control group I, II. Results5-FU concentration ratios for gloves and masks in exposed group I were significantly higher than those in exposed group II (P<0.05 or P<0.01). The average urinary 8-OHdG concentrations in exposed group I, control group I, exposed group II, and control group II were 14.69±0.93, 10.68±1.07, 10.57±0.55, and 11.96±0.73 ng/mg Cr, respectively. Urinary 8-OHdG concentration in exposed group I was significantly higher than that in control group I or that in exposed group II (P<0.01). There was a significant correlation between urinary 8-OHdG concentrations and spill frequencies per technician (P<0.01). ConclusionThere was detectable oxidative DNA damage in PIVAS technicians exposed to antineoplastic drugs. This oxidative DNA damage may be associated with their spill exposure experience and contamination of their personal protective equipment.

Pharmacokinetic Study of Weekly (Days 1-5) Low-dose S-1 in Patients with Non-Small-Cell Lung Cancer

S-1, an oral fluoropyrimidine, is usually given for 4 weeks (80 mg/m2/day) followed by a 2-week rest. However, compliance with this regimen is unsatisfactory because of adverse events such as leukopenia, anorexia, and nausea. To reduce adverse effects and improve compliance, we studied a "5-day on/2-day off" low-dose regimen of S-1 and evaluated pharmacokinetics in patients with non-small-cell lung cancer (NSCLC). Twelve patients with NSCLC were divided into 2 groups and received S-1 in a dose of 25 mg twice daily (level 1, n = 6) or 40 mg twice daily (level 2, n = 6) for 5 consecutive days followed by a 2-day rest (5 days on/2 days off) every week. Plasma 5-fluorouracil (5-FU) concentrations were measured. The maximum concentration in plasma and the area under the plasma concentration-time curve from 0 to 9 h were respectively 55.3 ± 21.1 ng/ml and 290.2 ± 95.7 ng·hr/ml for level 1, as compared with 104.2 ± 33.5 ng/ml and 541.9 ± 232.3 ng·hr/ml for level 2. These values were similar to those previously reported for a continuous intravenous infusion of 5-FU. Adverse events were grade 1 fatigue (n = 1 in each group) and anorexia (n = 1 in each group). A "5-day on/2-day off" low-dose (40 mg twice daily) regimen of S-1 is feasible for the treatment of NSCLC, with acceptable plasma 5-FU concentrations and minimal adverse effects. A phase II or III trial of this regimen in an adjuvant setting is warranted in patients with NSCLC.

The combination of hyperthermia or chemotherapy with gimeracil for effective radiosensitization

5-chloro-2,4-dihydroxypyridine (gimeracil) is a component of the oral fluoropyrimidine derivative S-1. Gimeracil was originally added to S-1 to yield prolonged 5-fluorouracil (5-FU) concentrations in serum and tumor tissues by inhibiting dihydropyrimidine dehydrogenase, which degrades 5-FU. We previously demonstrated that gimeracil enhances the efficacy of radiotherapy through the suppression of homologous recombination (HR) in DNA double strand repair. The goal of this paper was to examine the effects of gimeracil on the sensitivity of anticancer drugs and hyperthermia in order to obtain effective radiosensitization. Various cell lines, including DLD 1 (human colon carcinoma cells) and cells deficient in HR or nonhomologous end-joining (NHEJ), were used in clonogenic assays. The survival of these cells after various treatments (e.g., drug treatment, heat treatment, and radiation) was determined based on their colony-forming ability. Gimeracil enhanced cell-killing effects of camptothecin (CPT), 5-FU, and hydroxyurea. Gimeracil sensitized effects of CPT or 5-FU to cells deficient in HR or NHEJ to a similar extent as in other cells (DLD1 and a parent cell), indicating that its sensitizing mechanisms may be different from inhibition of HR or NHEJ. Combination of gimeracil and CPT or 5-FU sensitized radiation more effectively than each modality alone. Gimeracil also enhanced heat sensitivity at 42°C or more. The degree of heat sensitization with gimeracil increased as the temperature increased, and the combination of gimeracil and heat-sensitized radiation was more effective than each modality alone. Gimeracil enhanced sensitivity of CPT, 5-FU, and hyperthermia. Combination of these modalities sensitized radiation more efficiently than each modality alone.

The Effects of 5-fluorouracil Alone and in Combination with 13-cis Retinoic Acid and Vitamin D3 on Human Oral Squamous Cell Carcinoma Lines

Oral squamous cell carcinoma (OSCC) is responsible for about 90% of oral malignancies and its incidence is increasing. Despite various treatment protocols, survival rate of OSCC is low. Chemotherapy that is used for treating this carcinoma in advanced stages is systemic therapy that destroys carcinogenic cells, and controls tumor metastasis. Chemotherapy is very toxic and has limitations, especially for patients in advanced stages. Considering positive effects of retinoid and vitamin D3 derivatives in treating some carcinomas, we decided to evaluate the effect of combination of these drugs on OSCC. In this study the effects of combination of 5-fluorouracil, 13-cis retinoic acid and vitamin D3 on cultured cell of OSCC have been evaluated. OSCC cells were cultured in culture media and different concentration of 5-fluorouracil, 13-cis retinoic acid and vitamin D3 were added to cultured cell as separately and in combinations. The effect of treatment on cell proliferation and induction of apoptosis were evaluated by MTT and TUNEL assays respectively. Combination of 5-fluorouracil and 13- cis retinoic acid had the highest inhibitory effect on SCC cell proliferation. Combination of two drugs had more apoptotic effect than each of them separately, and combination of three drugs had more effect than combination of two drugs. Because combination of drugs had more inhibitory effect on cell proliferation than one of them and combination of three drugs had the most apoptotic effect than one of these drugs separately, these drugs may have synergic effect on OSCC. Combination of three drugs has more inhibitory effect on cell proliferation and apoptotic effect than one of these drugs.

Phase II study of S-1 monotherapy in patients with previously treated, advanced non-small-cell lung cancer

In this phase II clinical trial, we evaluated the efficacy and safety of S-1 monotherapy in patients with previously treated advanced non-small-cell lung cancer (NSCLC). We also measured plasma concentrations of 5-fluorouracil (5-FU) and 5-chloro-2,4-dihydroxypyridine components of S-1 and examined correlation with effectiveness and toxicity. S-1 was given orally at a dose of 80 mg/m(2)/day for 14 consecutive days, followed by a 7-day rest period. This treatment course was repeated until disease progression or intolerable toxicity. We enrolled 30 patients. The response rate was 26.7% (8/30), and the disease control rate was 70% (21/30). Median progression-free survival (PFS) was 3.1 months, and median overall survival (OS) was 11.2 months. Mutations in the epidermal growth factor receptor (EGFR) gene were analyzed in 27 patients. The response rate was higher in patients with mutant EGFR (50.0%) than in those with wild-type EGFR (11.8%, P = 0.0288). Median PFS was 4.8 and 2.5 months (P = 0.038), and median OS was 22.4 and 8.4 months (P = 0.071). There was no grade 4 toxicity in this study. Five patients had grade 3 non-hematologic toxicity, and there was a trend toward higher plasma concentrations of 5-FU in those patients than in another patients. S-1 monotherapy is effective and well-tolerated treatment for previously treated advanced NSCLC.

USING ULTRAFILTRATION TO FACILITATE SIMULTANEOUS QUANTIFICATION OF 5-FLUOROURACIL IN MOUSE PLASMA AND TISSUES BY HPLC

A simple and rapid HPLC method that includes ultrafiltration to remove plasma and tissue interference component was developed to determine 5-Fluorouracil (5-FU) concentration in mouse plasma and tissues (heart, liver, spleen, lung, and kidney). Following ultrafiltration, standards, quality controls, and plasma samples were separated by reversed-phase HPLC on an ODS analytical column and quantified using UV detection at 265 nm. The mobile phase was 5 mM phosphate buffer (pH 5)-methanol (95:5, v/v), with a flow-rate of 0.6 mL/min. The calibration curves for 5-FU in mouse plasma and tissue samples were all linear (r2 > 0.997) over the drug concentration range of 40 to 25000 ng/mL. For all plasma and tissue samples, both the intra- and inter-day precisions were less than 5% expressed in RSD, and the accuracy was between 96 and 104%. The limit of detection was 20 ng/mL. The overall recoveries were >73%. The method was illustrated with a preliminary in vivo analysis on 3 mice treated with intravenous injection of 5-FU, and most of the drug was found deposited in mouse liver 1 hour after the administration.

Effects of plasma concentrations of 5-fluorouracil on long-term survival after treatment with a definitive 5-fluorouracil/cisplatin-based chemoradiotherapy in Japanese patients with esophageal squamous cell carcinoma

BackgroundA substantial body of literature has accumulated during the past 20 years showing the plasma concentrations of 5-fluorouracil (5-FU) to correlate with clinical response and/or toxicity in colorectal cancer, and head and neck cancer, but little information is available concerning effects on long-term survival. Here, Japanese patients with esophageal squamous cell carcinoma (ESCC) were followed up for 5 years after treatment with a definitive 5-FU/cisplatin (CDDP)-based chemoradiotherapy (CRT), and the association between prognosis and the plasma concentration of 5-FU was evaluated.MethodsForty-nine patients with ESCC, who were treated with a definitive 5-FU/CDDP-based CRT, were enrolled. A course consisted of the continuous infusion of 5-FU at 400 mg/m2/day for days 1-5 and 8-12, the infusion of CDDP at 40 mg/m2/day on days 1 and 8, and the radiation at 2 Gy/day on days 1 to 5, 8 to 12, and 15 to 19, with a second course repeated after a 2-week interval. Plasma concentrations of 5-FU were determined by high performance liquid chromatography at 5:00 PM on days 3, 10, 38 and 45, and at 5:00 AM on days 4, 11, 39 and 46.ResultsThe overall 5-year survival rate was 42.9%. Age (P = 0.020), body weight (P = 0.019), and disease stage (P = 0.048) affected the survival, and the survival depended on the clinical response assessed at 1 month after the treatment (P = 0.001). Higher plasma concentrations of 5-FU resulted in a better clinical response (P = 0.043), and trended to prolong survival (P = 0.321).ConclusionsThe long-term survival after treatment with a definitive 5-FU/CDDP-based CRT possibly depends on the plasma concentrations of 5-FU, and further clinical studies with a larger number of cases are needed to clarify the relationship between them.

Screening of anticancer drugs for chemoembolization of hepatocellular carcinoma

The aim of this study was to select the best candidate drug for transarterial chemoembolization by in-vitro cytotoxic evaluations of 11 anticancer drugs on three human hepatocellular carcinoma (HCC) cell lines. The SNU-398, HepG2, and SNU-449 human HCC cell lines were exposed for 30 min to 11 concentrations of doxorubicin, epirubicin, idarubicin, mitoxantrone, carboplatin, cisplatin, oxaliplatin, 5-fluorouracil, gemcitabine, mitomycin C, or paclitaxel. Cytotoxicity was measured using a quantitative colorimetric assay. For each drug and cell line, we calculated the drug concentration that caused 90% cell death (IC90). To enable comparisons of drugs with different concentration ranges, we computed the cytotoxic index (CyI) as the ratio of maximal drug concentration of more than IC90. Parameters were estimated using nonlinear regression models. Idarubicin was the most active drug on all three cell lines. With SNU-398 cells, the idarubicin CyI was 2.4-fold, 2.5-fold, 57-fold, 148-fold, and more than 58 748-fold higher than the CyIs of mitoxantrone, epirubicin, doxorubicin, gemcitabine, and other drugs, respectively. With HepG2 cells, the idarubicin CyI was 27-fold, 28-fold, 51-fold, and more than 1343-fold higher than the CyIs of doxorubicin, epirubicin, mitoxantrone, and other drugs, respectively. On the resistant SNU-449 cell line, the idarubicin CyI was 2.9-fold and 14-fold higher than the CyIs of paclitaxel and gemcitabine, respectively, the only other drugs effective on this cell line. Among 11 chemotherapeutic agents including doxorubicin, cisplatin, and epirubicin, the most effective on three HCC cell lines was idarubicin. Further clinical investigations are needed to evaluate the safety and efficacy of idarubicin for transarterial chemoembolization in HCC.

Gimeracil, an inhibitor of dihydropyrimidine dehydrogenase, inhibits the early step in homologous recombination

Gimeracil (5-chloro-2, 4-dihydroxypyridine) is an inhibitor of dihydropyrimidine dehydrogenase (DPYD), which degrades pyrimidine including 5-fluorouracil in the blood. Gimeracil was originally added to an oral fluoropyrimidine derivative S-1 to yield prolonged 5-fluorouracil concentrations in serum and tumor tissues. We have already reported that gimeracil had radiosensitizing effects by partially inhibiting homologous recombination (HR) in the repair of DNA double strand breaks. We investigated the mechanisms of gimeracil radiosensitization. Comet assay and radiation-induced focus formation of various kinds of proteins involved in HR was carried out. siRNA for DPYD were transfected to HeLa cells to investigate the target protein for radiosensitization with gimeracil. SCneo assay was carried out to examine whether DPYD depletion by siRNA inhibited HR repair of DNA double strand breaks. Tail moments in neutral comet assay increased in gimeracil-treated cells. Gimeracil restrained the formation of foci of Rad51 and replication protein A (RPA), whereas it increased the number of foci of Nbs1, Mre11, Rad50, and FancD2. When HeLa cells were transfected with the DPYD siRNA before irradiation, the cells became more radiosensitive. The degree of radiosensitization by transfection of DPYD siRNA was similar to that of gimeracil. Gimeracil did not sensitize DPYD-depleted cells. Depletion of DPYD by siRNA significantly reduced the frequency of neopositive clones in SCneo assay. Gimeracil partially inhibits the early step in HR. It was found that DPYD is the target protein for radiosensitization by gimeracil. The inhibitors of DPYD, such as gimeracil, could enhance the efficacy of radiotherapy through partial suppression of HR-mediated DNA repair.

The Effect of Fluorouracil on the Transepithelial Electrical Resistance of Caco-2 Cells Growing as Confluent Monolayers on Polycarbonate Filters

In a previous study we showed that enteric coated mucoadhesive particles administered orally delivered 5-fluorouracil slowly to the colon. This present study extends these investigations into an in-vitro cell line. Human colonic adenocarcinoma cells (Caco-2) were cultured as confluent monolayers on polycarbonate filters in Transwell systems for investigating in-vitro cytotoxic effects of different concentrations of 5-fluorouracil over the range 0.077-7.7 μM. Cell damage was determined by measuring changes in the transepithelial electrical resistance (TEER). The results suggested that 5-fluorouracil at all concentrations tested significantly affected the Caco-2 cells. Application of residuals to the experimental TEER data indicated that the relationship between square-root of time and TEER was linear (P > 0.05), indicating that the drug may affect the Caco-2 cells by a diffusional mechanism. There was no suggestion of a minimal or threshold concentration of drug required to affect the cells over the range tested.