High-throughput method to analyze tegafur and 5-fluorouracil in human tears and plasma using hydrophilic interaction liquid chromatography/tandem mass spectrometry.

RATIONALE: We developed a new high-throughput method to analyze tegafur (FT) and 5-fluorouracil (5-FU) in tear and plasma samples using hydrophilic interaction liquid chromatography (HILIC)/tandem mass spectrometry (MS/MS). METHODS: The tear samples (10 µL) spiked with FT, 5-FU, and 5-chlorouracil (internal standard) were diluted using 40 µL of 2 M ammonium acetate and 250 µL of acetonitrile with 2% formic acid; 20 µL of plasma spiked with the two drugs and internal standard was diluted with 80 µL of 2 M ammonium acetate and 500 µL of acetonitrile with 2% formic acid. After centrifugation, the clear supernatant extract (15 µL) was directly injected into the HILIC/MS/MS instrument, and each drug was separated on a Unison UK-Amino column (50 mm × 3 mm i.d., 3 µm particle size) with a linear gradient elution system composed of 10 mM ammonium acetate (pH 6.8) and acetonitrile at a flow rate of 0.7 mL/min. We performed quantification by multiple reaction monitoring (MRM) with negative-ion atmospheric-pressure chemical ionization. RESULTS: Distinct peaks were observed for the drugs on each MRM channel within 2 min. The regression equations showed good linearity within the range 0.04-4.0 µg/mL for the tear and plasma samples with detection limits at 0.02-0.04 µg/mL. Recoveries for target analytes (FT and 5-FU) for the tear and plasma samples were in the 94-128% and 94-104% ranges, respectively. The intra- and inter-day coefficients of variation for the two drugs were lower than 10.8%. The accuracies of quantitation were 97-115% for both samples. CONCLUSIONS: We established a high-throughput, reproducible, and practical procedure for analyzing FT and 5-FU in human tear and plasma samples using HILIC/MS/MS analysis with an aminopropyl-bonded mixed-mode separation column. This method can be applied to the high-throughput routines used in clinical analyses.

Pharmacokinetics of S-1 monotherapy in plasma and in tears for gastric cancer patients.

BACKGROUND: S-1 is an oral anticancer drug composed of tegafur (FT), which is a prodrug of 5-FU, 5-chloro-2,4-dihydroxypyridine (CDHP), and potassium oxonate. Recently, some studies have been reported on watering eyes caused by S-1. However, the mechanism of watering eyes caused by S-1 is still unclear. The aim of this study was to investigate the correlation between tears and plasma concentrations of FT, 5-FU, and CDHP, which are components and active modulator of S-1. METHODS: We prospectively investigated the pharmacokinetics (PK) of FT, 5-FU, and CDHP in plasma and in tears of gastric cancer patients who were treated with S-1 monotherapy at the dose of 80 mg/m2/day. Plasma and tears from both eyes were obtained 1, 2, 4, and 8 h after S-1 administration on day 1 and 14 of the first cycle. RESULTS: Total of eight patients were enrolled. All the FT, 5-FU and CDHP were detected both in plasma and in tears, and their PK parameters were measured. There was a positive correlation between the concentrations of FT, 5-FU and CDHP in the plasma and those in the tears on day 1 and day 14 (correlation coefficients r, right eye/left eye: r = 0.882/0.878, 0.877/0.890, and 0.885/0.878, respectively). CONCLUSION: There was a positive correlation between the concentrations of FT, 5-FU and CDHP in the plasma and those in the tears. The result is expected to facilitate the further investigation into the causes of watering eyes and the establishment of the effective methods for the prevention and the treatment.

A review of analytical methods for the determination of 5-fluorouracil in biological matrices.

5-Fluorouracil (5-FU) is a cytostatic agent that has been widely used in the treatment of various solid tumours for more than 20 years, and is still considered to be among the most active antineoplastic agents in advanced colorectal cancer and malignancies of the head and neck. A large number of non-chromatographic and chromatographic methods for the quantitation of 5-FU, related prodrugs and their metabolites in biological matrices have been developed in the last 30 years to support preclinical and clinical studies. However, 5-FU monitoring has not been widely used, at least not in the USA, and certainly not outside the clinical research setting, given the absence of simple, fast and inexpensive testing methods for 5-FU monitoring. Recent developments with testing based on liquid chromatography-tandem mass spectrometry and a nanoparticle antibody-based immunoassay may facilitate routine monitoring of 5-FU in daily clinical practice. In this review the advantages and disadvantages of the bioanalytical methods developed and used for 5-FU, its metabolites and related prodrugs are discussed.

Stoichiometric molecularly imprinted polymers for the recognition of anti-cancer pro-drug tegafur.

Molecularly imprinted polymers (MIPs) targeting tegafur, an anti-cancer 5-fluorouracil pro-drug, have been prepared by stoichiometric imprinting using 2,6-bis(acrylamido)pyridine (BAAPy) as the functional monomer. Solution association between tegafur and BAAPy was studied by (1)H NMR titration, which confirmed the formation of 1:1 complexes with an affinity constant of 574±15M(-1) in CDCl3. Evaluation of the synthesised materials by HPLC and equilibrium rebinding experiments revealed high selectivity of the imprinted polymer for the pro-drug vs. 5-fluorouracil and other competing analytes, with maximum imprinting factors of 25.3 and a binding capacity of 45.1µmolg(-1). The synthesised imprinted polymer was employed in solid-phase extraction of the pro-drug using an optimised protocol that included a simple wash with the porogen used in the preparation of the material. Tegafur recoveries of up to 96% were achieved from aqueous samples and 92% from urine samples spiked with the template and three competing analytes. The results demonstrate the potential of the prepared polymers in the pre-concentration of tegafur from biological samples, which could be an invaluable tool in the monitoring of patient compliance and drug uptake and excretion.

Pharmacokinetics of S-1, an oral formulation of ftorafur, oxonic acid and 5-chloro-2,4-dihydroxypyridine (molar ratio 1:0.4:1) in patients with solid tumors.

S-1 is an oral formulation of ftorafur (FT), oxonic acid and 5-chloro-2,4-dihydroxypyridine (CDHP) at a molar ratio of 1:0.4:1. FT is a 5-fluorouracil (5-FU) prodrug, CDHP is a dihydropyrimidine dehydrogenase (DPD) inhibitor and oxonic acid is an inhibitor of 5-FU phosphoribosylation in the gastrointestinal mucosa and was included to prevent gastrointestinal toxicity. We determined the pharmacokinetics of S-1 in 28 patients at doses of 25, 35, 40 and 45 mg/m(2). The plasma C(max) values of FT, 5-FU, oxonic acid and CDHP increased dose-dependently and after 1-2 h were in the ranges 5.8-13 microM, 0.4-2.4 microM, 0.026-1.337 microM, and 1.1-3.6 microM, respectively. Uracil levels, indicative of DPD inhibition, also increased dose-dependently from basal levels of 0.03-0.25 microM to 3.6-9.4 microM after 2-4 h, and 0.09-0.9 microM was still present after 24 h. The pharmacokinetics of CDHP and uracil were linear over the dose range. The areas under the plasma concentration curves (AUC) for CDHP and uracil were in the ranges 418-1735 and 2281-8627 micromol x min/l, respectively. The t(1/2) values were in the ranges 213-692 and 216-354 min, respectively. Cumulative urinary excretion of FT was predominantly as 5-FU and was 2.2-11.9%; the urinary excretion of both fluoro-beta-alanine and uracil was generally maximal between 6 and 18 h. During 28-day courses with twice-daily S-1 administration, 5-FU and uracil generally increased. Before each intake of S-1, 5-FU varied between 0.5 and 1 microM and uracil was in the micromolar range (up to 7 microM), indicating that effective DPD inhibition was maintained during the course. In a biopsy of an esophageal adenocarcinoma metastasis that had regressed, thymidylate synthase, the target of 5-FU, was inhibited 50%, but increased four- to tenfold after relapse in subsequent biopsies. In conclusion, oral S-1 administration resulted in prolonged exposure to micromolar 5-FU concentrations due to DPD inhibition, and the decrease in uracil levels after 6 h followed the pattern of CDHP and indicates reversible DPD inhibition.

1-(Tetrahydro-2-furanyl)-5-fluorouracil (Ftorafur) determined in rat hair as an index of drug exposure.

The fluorescence derivatization of 1-(tetrahydro-2-furanyl)-5-fluorouracil (Ftorafur, FT) with 4-bromomethyl-6,7-dimethoxycoumarin (BrMdmc) with 18-crown-6 as catalyst was utilized for a sensitive and selective liquid chromatographic method to determine the concentration of FT in hair. Hair samples collected from rats, which had received FT intraperitoneally for 1 to 4 weeks, were dissolved in 1 M NaOH by heating at 80 degrees C for 30 min. A three-step extraction procedure for FT in the dissolved hair was used before and after the derivatization of FT with BrMdmc. The detection limit achieved was < 0.16 ng/mg hair. A dose-dependent accumulation of FT was evident in the rat hair (r = 0.914, p < 0.001): 0.24 +/- 0.07, 1.35 +/- 0.39, and 2.85 +/- 0.74 ng/mg hair (mean +/- SD, n = 4-5) at the doses of 5, 15, and 50 mg/kg/day for 4 weeks, respectively. In the rats that had received the highest dose of FT, the accumulation of FT in the hair depended also on the duration of FT administration: 0.82 +/- 0.33 (1 week), 1.52 +/- 0.38 (2 weeks), and 2.85 +/- 0.74 (4 weeks) ng/mg hair (n = 4-5). These findings suggest that the analysis of an oral antineoplasmic FT in the hair may be useful for assessing the degree of exposure to the drug.

GLC determination for ftorafur in biological fluids.

A GLC analysis for free ftorafur was developed to follow in drug disposition in body fluids of patients. The free drug was extracted from aqueous biological samples with chloroform, derivatized by methylation, and chromatographed on 1% HI-EFF 8BP using flame-ionization detection. The analysis is sensitive (0.25 microgram/ml of plasma) and specific for the intact molecule, and it does not interfere with subsequent fluorouracil analysis of the same sample.

High-pressure liquid chromatographic determination of ftorafur [1-(tetrahydro-2-furanyl)-5-fluorouracil] and GLC-mass spectrometric determination of 5-fluorouracil and uracil in biological materials after oral administration of uracil plus ftorafur.

Oral administration of uracil plus ftorafur [1-(tetrahydro-2-furanyl)-5-fluorouracil] has a greater antitumor effect than that of ftorafur alone. A high-pressure liquid chromatographic methods was developed for the determination of ftorafur in plasma and visceral tissues with a sensitivity of 0.025 microgram/ml or g of wet weight. A combination of GLC--mass fragmentography and GLC--mass spectrometry with total-ion monitoring was developed for the specific, simultaneous determination of 5-fluorouracil, and active metabolite, and uracil as their trimethylsilylated derivatives. The detection limits for 5-fluorouracil and uracil in the first method were 0.001 microgram/ml for plasma and 0.001--0.005 microgram/ml for visceral tissues, and those in the second method were 0.2 microgram/ml or g of wet weight. The precision and sensitivity of the assay appear to be satisfactory for determination of the levels of these compounds in plasma and visceral tissues.

Determination of the 5-fluorouracil and N1(2'-furanidyl)uracil in the presence of tegafur by zero-crossing first derivative spectrometry.

A first derivative spectrometric method has been developed for the determination of the 5-fluorouracil and N1(2'-furanidyl)uracil related substances and degradation products of tegafur. The wavelengths selected for the determination of 5-fluorouracil and N1(2'-furanidyl)uracil were 298 and 288 nm, respectively. At this wavelength, the calibration graphs between the amplitude of the signals and the concentration of each compound were linear up to 24.75 mg l(-1) for 5-fluorouracil and up to 20.20 mg l(-1) for N1(2'-furanidyl)uracil. The detection limits were 0.40 and 0.050 mg l(-1) for 5-fluorouracil and N1(2'-furanidyl)uracil, respectively. The method is simple and rapid and does not require any preliminary treatment of the sample. The method was validated.

Concentration of 5-fluorouracil in renal cells from cancer patients administered a mixture of 1-(2-tetrahydrofuryl)-5-fluorouracil and uracil.

The concentrations of 5-fluorouracil (5-FU) and related compounds were studied in tissues including renal cells removed from 27 cancer patients, to whom UFT (a mixture of uracil and tegafur in a molecular ratio of 4:1) was administered orally over 5 days. The level of 5-FU in cancer tissue was two-fold that of the normal kidney (87 +/- 79 ng/g vs. 38 +/- 29 ng/g, p less than 0.005), with almost the same level of tegafur. The serum 5-FU level was as low as 4 ng/ml. These results confirm the high efficiency of UFT with minimal side-effects compared to tegafur.

[Thymidylate synthase inhibition and concentration of 5-fluorouracil after administration of UFT in human uroepithelial carcinomas].

Inhibition of thymidylate synthase (TS) and the concentration of tegafur, 5-FU and uracil in the tumor and the non-tumor tissues were compared in 18 uroepithelial cancer patients who had been administered UFT (600 mg/day) for seven days before operation. 5-FU and uracil levels in the tumor tissue were increased to 5.1 and 3.6 fold, respectively, compared those in the normal tissue, although there was no difference in tegafur levels between normal and tumor tissue. The mean inhibition rate of TS activity in the tumor tissue was significantly higher (36%) than that in the normal tissue (21%). However, no correlation between 5-FU level and inhibition rate of TS activity was found in either tissue. Not only the higher tumor concentration of 5-FU but also the higher inhibition of TS activity in the tumor tissue suggests that UFT is likely a useful drug for the treatment of uroepithelial carcinomas.

[Preoperative treatment of FT-207 suppository in cervical cancer-- serum and tumor tissue content of FT-207].

A 750 mg FT suppository was inserted daily for seven days prior to surgery of uterine cervical carcinoma. The concentrations of FT and 5-FU in the serum, tumor tissue, adjacent normal tissues and regional lymph nodes were then measured. In addition, the concentrations of FT and 5-FU in the serum of patients who had been receiving chemotherapy for long term (an average of 15 months) after the initial remission were measured. The results are as follows: The 5-FU concentration in the serum of patients following short duration of administration was 0.014 +/- 0.006 micrograms/micromilligram. The 5-FU concentration in the tumor tissue was 0.209 +/- 0.132 microgram/g. This was approximately 3.2 times higher than the concentration in the normal tissue which was 0.065 +/- 0.017 microgram/g, and approximately 2.4 times higher than in the regional lymph nodes of 0.088 +/- 0.055 microgram/g. Relatively higher concentrations of 5-FU were seen in the non-keratinizing type than in the keratinizing type suggesting. The correlation between the concentration in the tumor tissue and the histologic findings of the carcinoma tissue. The 5-FU concentration in the serum of patients receiving long-term chemotherapy was 0.019 +/- 0.015 microgram/micromilligram, showing no significant difference from the patients receiving short-term chemotherapy.

[Antitumor effect of UFT on human ovarian cancer grafted to nude mice and 5-FU concentration in tumor and normal tissues].

Antitumor effects of UFT, tegafur (FT-207), cisplatin (CDDP) and the combination of UFT with CDDP on a human ovarian cancer xenograft in nude mice and the concentration of 5-FU in the tumor tissue and major organs were studied. UFT (48.6mg/kg/day X 20) or tegafur (15.0mg/kg/day X 20) was daily administrated orally, and CDDP (5mg/kg/day X 3) was administrated intraperitoneally at an 7-day interval. The inhibition rates of the tumor growths were 49.6% with UFT, -2.3% with tegafur and 17.7% with CDDP, respectively. In the combination of UFT with CDDP, severe side effect were observed. The concentration of 5-FU in UFT-treated group was higher than tegafur group: about 2 times in the tumor, 5 times in the liver, 9 times in the kidney and 4 times in the spleen, respectively. In the combination of UFT with CDDP, the concentration of 5-FU in major organs, especially in the kidney, in nude mice that died at 10 day after drug administration were higher than in those of UFT. These findings indicate that UFT increases the intratumoral concentration of 5-FU to elicit better antitumor effect and also the concentration of 5-FU in various normal organs after long time administration.

[Blood and tumor levels of tegafur, 5-fluorouracil, and uracil].

The blood and tissue concentrations of tegafur (TGF), 5-fluorouracil (5-FU) and uracil were tested by administering 6 capsules of UFT (600 mg as tegafur (TGF)) per day to 21 patients awaiting operation for stomach cancer. At the same time, comparative tests were performed by dividing the patients into two groups, giving UFT before meals in one group and in another after meals. Both 5-FU and uracil levels tend to reach to the maximum blood concentration after one hour, while showing similar variations with time lapse. Furthermore, a significantly positive relationship was observed between 5-FU and uracil in the rate of changes of blood concentration during six hours after administration of UFT. The 5-FU concentration within tumor tissue was 10 times higher than that within the blood and 2 times higher than that within normal tissue. A similar tendency was observed with uracil, which was of value in the clinical determination of the effect of uracil in inhibiting decomposition of 5-FU. With regard to the comparative test between the groups of pre- and post-meal UFT administration, the concentration of tegafur (TGF) and 5-FU in tumor tissue tended to be higher in the pre-meal group which indicates a need for further study of the phenomenon.

[Level of 5-FU in tumor tissues of the patients with head and neck malignant tumors after oral administration of UFT].

Levels of futraful (FT-207), 5-FU and Uracil in blood and tissue were measured on 11 cases of oral malignant tumors after administration of UFT. 1) After administration of UFT, the highest level of 5-FU was detected in tumor tissue followed by the normal lymph node, metastatic lymph node, and normal tissue which showed the lowest level. 2) Levels of 5-FU in serum was lower than in tumor, and T/S ratio of levels of 5-FU was 9,190. 3) There were significant regressions in levels of 5-FU and Uracil in normal lymph node (p less than 0.02). Coefficient of correlation was 0.469. 4) Comparisons of levels of 5-FU after administration of UFT and futraful were 1.89 in tumor, 1.72 in normal tissue, 1.80 in metastatic lymph node and 1.55 in normal lymph node. Level of 5-FU after administration of UFT was higher than that of futraful. 5) Higher level of 5-FU after administration of UFT in tumor tissue and lymph node were maintained for a longer period.

[Study on the concentration of FT-207 and 5-FU in serum and tissues of bladder tumor and prostatic carcinoma].

Serum and tissue concentrations of 5-FU and FT-207 were estimated in 26 patients with bladder tumor or prostatic carcinoma after administration of FT-207 suppositories. The 5-FU concentration in bladder tumor was higher than in normal mucosal bladder tissue. The 5-FU concentration of prostatic cancer was almost equal to that of normal prostatic tissue. Relatively higher concentrations of 5-FU were recognized in bladder tumor of a high stage than of a low stage. There were no differences of serum and tissue 5-FU concentration in bladder tumor between patients more than 65 years old and those under 65 years old. No side-effects occurred in any case during suppository administration.

[Serum and tissue concentrations of UFT in patients with lung cancer].

Postoperative serum and tissue concentrations of 5-FU, FT-207 and uracil were measured in 36 patients with lung cancer who were administered UFT for seven days preoperatively. The concentration of 5-FU was high in tumor tissue and lymph nodes, but very low in serum. Such differences were not observed in the FT-207 levels. Tumor concentration of 5-FU in patients administered daily doses of 600 mg was 0.151 +/- 0.099 microgram/g which was three times higher than the minimum inhibitory concentration, and higher than that seen with other doses. The histological type and T factor were not related to the tissue concentration of 5-FU. Lymph node metastasis was not related to the concentration of 5-FU in the lymph nodes. The optimal daily dose of UFT for patients with lung cancer was considered to be 600 mg.

[Evaluation of tissue concentrations of FT-207 and 5-FU in head and neck cancer].

FT-207 was administered to 30 patients with squamous cell carcinoma of the head and neck. After FT-207 administration tumor concentration of FT-207 and 5-FU was measured using a chemical assay method. Differences of FT-207 and 5-FU in total doses, administration routes (oral, intrarectal, and intravenous), tumor sites (larynx, maxillary sinus, tongue, neck and hypopharynx), and types of differentiation (well-differentiated, moderately-differentiated, and poorly-differentiated) were studied and the following results were obtained: 1. In FT-207 tumor concentration study, no significant difference was observed in any group. 2. 5-FU tumor concentration increased in higher total administration doses and in advanced differentiated type groups as well as in the non-irradiated group. 3. 5-FU tumor concentration was measured by T/N ratio; oral and intravenous administration methods showed 4.5 (P less than 0.05) and 2.4 (P less than 0.10) respectively. In larynx tumor the highest concentration of 3.9 (P less than 0.05) was obtained among various tumors. The value of 4.0 (P less than 0.05) was yielded in the well-differentiated type, which was significantly higher compared to that of other differentiation types. Overall T/N ratio (5-FU concentration in the tumor/5-FU concentration in the normal tissue) was 2.7 (P less than 0.025).

[The serum and tissue concentrations of tegafur, 5-fluorouracil and uracil in patients with gastric and colorectal cancers after oral administration of UFT].

Fifteen patients with stomach cancer and colorectal cancer were orally administered 600 mg of UFT before operation and the concentration of tegafur, 5-fluorouracil (5-FU) and uracil in the tissues and serum were measured by chemical assay. The mean 5-FU levels in tumor and lymph node were higher than those in normal tissues and were maintained in tumor for long time. Uracil level in tumor tissue was higher than in normal tissue with or without administration of UFT. Positive correlation between the concentration of uracil and 5-FU in the colorectal tumor with the correlation coefficient of 0.87 (p less than 0.01) was obtained.

[Study on the pharmacokinetics of UFT in patients with hepatocellular carcinoma associated with cirrhosis].

UFT was administered preoperatively in 20 cases of primary hepatocellular carcinoma, and tegafur, 5-fluorouracil (5-FU), uracil, total thymidylate synthase (TS) and free TS in blood and liver tissue were determined. The results were as follows. 1. Tegafur level was significantly high in blood, while the levels of 5-FU and uracil were high in the liver tissue. The 5-FU level in the cancerous area was 0.099 micrograms/g, higher than the effective level regardless of the presence or not of complication with liver cirrhosis. 2. Total TS, FdUMP (computed as total TS-free TS) and TS inhibition rate [(computed as (TS-free TS)/TS x 100 (%)] were significantly higher in the cancerous liver tissue than in the non-cancerous area. In the cases where determination was made simultaneously for the cancerous liver and non-cancerous liver tissues, the FdUMP level and TS inhibition rate in the cancerous liver tissue were high in 14 out of 17 cases (82.4%) and 13 out of 17 cases (76.5%) respectively. Therefore, UFT seems to have selective toxicity. 3. No correlation was found between 5-FU level, total TS, FdUMP in the liver tissue on the one hand and TS inhibition rate on the other. 4. UFT yielded sufficient 5-FU levels in the cancerous area regardless of the presence or not of complication with cirrhosis and therefore is expected to inhibit DNA synthesis selectively in the cancerous tissue.