Penetration of capecitabine and its metabolites into malignant and healthy tissues of patients with advanced breast cancer.

Capecitabine is an oral prodrug of 5-fluorouracil (FU). Since FU concentrations achieved in malignant lesions are an important determinant of efficacy, we investigated the intratumoral transcapillary transfer of capecitabine and its metabolites in vivo. A total of 10 patients with skin metastases from breast cancer received a daily dose of 2500 mg m(-2) capecitabine administered orally in two divided doses for 2 weeks. Microdialysis probes were inserted into a cutaneous metastasis and subcutaneous connective tissue to evaluate the interstitial tissue pharmacokinetics of capecitabine and its metabolites 5'-deoxy-5-fluorocytidine (DFCR), 5'-deoxy-5-fluorouridine (DFUR), and FU by capillary electrophoresis. As intended with the prodrug design of capecitabine, FU was present in low concentrations in tumour interstitium (median c(max): 0.26 microg ml(-1)) when compared with capecitabine, DFCR, and DFUR (median c(max): 2.66, 4.22, and 2.13 microg ml(-1), respectively). Capecitabine and its metabolites easily penetrated malignant and healthy tissue and equilibrated within 45 min between plasma and tissue interstitium. Considering tissue exposure at the extracellular level, no significant differences between healthy and malignant tissues were observed. Our data show that absorption and metabolism determined the tissue pharmacokinetics of capecitabine. There was no evidence of drug tolerance, which may be attributed to impaired transcapillary transfer into tissue, even after repeated administration as shown for three patients.

On-line solid-phase extraction and determination of paclitaxel in human plasma.

The application of coupled-column liquid chromatographic analysis to pharmacokinetic studies eliminates the need for sample clean-up from plasma. Considering lipophilic antineoplastic agents, we tested this approach to analyze paclitaxel under unfavourable circumstances (i.e., weekly low-dose regimen, plasma protein binding >90%, UV detection at 229 nm). The excellent quality control data (recovery: 95.6-100.7%, inter-assay relative standard deviation on 5 days: 1.3-3.2%, accuracy: 0.9-2.7%) and the detection limit of 19 nM indicates the usefulness of this method for the analysis of paclitaxel in plasma using on-line solid-phase extraction.

Penetration of dacarbazine and its active metabolite 5-aminoimidazole-4-carboxamide into cutaneous metastases of human malignant melanoma.

BACKGROUND: Dacarbazine has been on the market for approximately 3 decades but remains the most effective single agent available for the therapy of metastatic malignant melanoma (MMM). Most MMMs, however, respond poorly to dacarbazine therapy. Apart from tumor resistance at a molecular level, several studies support the notion that therapeutic failure in tumor therapy also might be attributed to an impaired transcapillary drug transfer. METHODS: On the basis of this hypothesis, the authors measured intratumor transcapillary transfer rates of dacarbazine and its active metabolite 5-aminoimidazole-4-carboxamide (AIC) by in vivo microdialysis after intravenous administration of dacarbazine at doses of 200 mg/m(2) to 1000 mg/m(2) (n = 7) in patients suffering from MMM. RESULTS: For all doses, area under the concentration curve (AUC) values for dacarbazine and AIC were not significantly different between plasma and tumor interstitium with AUC(tumor)/AUC(plasma) ratios of 0.97 +/- 0.08 (mean +/- standard error of the mean) for dacarbazine and 0.76 +/- 0.22 for AIC. AUC(0-240) values for dacarbazine and AIC measured in plasma correlated closely with corresponding AUC(0-240)values measured in the interstitium of MMMs with values of r(s) = 0.82 (P = 0.042) and r(s) = 0.90 (P = 0.037), respectively. CONCLUSIONS: The results of this study indicate favorable tumor penetration characteristics of dacarbazine and its active metabolite AIC. The relative lack of response to antineoplastic therapy with dacarbazine, thus might be explained by resistance of melanoma cells at a molecular level rather than by an inability of dacarbazine and AIC to penetrate into the interstitium of MMM.

Reverse transcriptase template switching during reverse transcriptase-polymerase chain reaction: artificial generation of deletions in ribonucleotide reductase mRNA.

Using reverse transcriptase-polymerase chain reaction (RT-PCR), we have recently described a bona fide deletion within the coding sequence of the large subunit of ribonucleotide reductase (R1) mRNA in colon cancer. Consecutive studies have raised questions about the nature of this phenomenon, because the corresponding genomic alteration at the DNA level or an aberrant protein could not be detected. Thus we considered an in vitro artifact during RT-PCR as a possible explanation for this observation. In contrast to reverse transcriptase, Taq DNA polymerase or C. therm DNA polymerase did not generate the aberrant product, suggesting the demand for the template switching activity intrinsic to retroviral reverse transcriptases. In fact, virtually the same deletion was observed in RT-PCR experiments when in vitro transcribed R1 mRNA was used. Considering structural prerequisites for template switching within R1 mRNA, we show that two direct repeats adjacent to a strong stem-loop secondary structure flank the deleted region of 1851 base pairs. Because several mRNAs encoding proteins of clinical and diagnostic importance fulfill these criteria, template switching enhances the potential risk of observing artifacts when interpreting results from RT-PCR studies. As shown in the present example, this may involve the artificial generation and the misinterpretation of PCR fragments amplified from targets relevant to tumor biology or cancer pharmacology. As a possible solution, one-step PCR with C. therm polymerase should be considered. This polymerase eliminates the artificial generation of aberrant mRNA signals observed during cDNA synthesis.

Long-term monitoring of sister chromatid exchanges and micronucleus frequencies in pharmacy personnel occupationally exposed to cytostatic drugs.

OBJECTIVES: Many antineoplastic drugs were found to have carcinogenic, mutagenic and teratogenic potential. The aim of this study was to carry out cytogenetic and internal dose monitoring of hospital pharmacy personnel regularly involved in the preparation of cytostatic agents, in order to test possible cytostatics-induced genotoxic effects due to occupational exposure under routine working conditions, and in cases of accidental contamination. METHODS: Platinum in whole blood and anthracyclines in plasma were measured to assess internal exposure to cytostatics. The level of cytogenetic damage was determined in peripheral blood lymphocytes with the micronucleus test and the sister chromatid exchange assay. Five series of monitoring were performed over a period of 2 years. RESULTS: No significant differences in the mean frequencies of sister chromatid exchanges (SCE) and micronuclei (MN) were found between occupationally exposed probands and controls (9.9 +/- 1.4 vs 10.1 +/- 1.2 SCEs/cell and 21.2 +/- 7.2 vs 23.3 +/- 7.5 MN/2000 binucleated (BN) cells, n = 16). Significant elevations of SCE or MN were detected in seven out of 12 cases of accidental contamination at the workplace, whereas no increase in platinum in blood and anthracyclines in plasma was observed in these probands. Two cases of non-reported contamination were identified by measurement of epirubicin in plasma. Smoking was found to increase the SCE significantly. No correlation between individual SCE scores and MN scores was observed. CONCLUSIONS: Our findings support a transient increase in SCE or MN after relevant exposure to cytostatic drugs in cases of accidental contamination. The lack of significant differences in SCE and MN between hospital pharmacy personnel and unexposed controls, points to high standards of safety at the corresponding workplaces.

Negative chronotropic effects of fentanyl attenuate beneficial effects of dobutamine on oxygen metabolism: hemodynamic and pharmacokinetic interactions.

Opioids are well known to cause cardiovascular depression. The aim of the present investigation was to determine whether an interaction of opioid derivatives with catecholamines might be involved in these hemodynamic alterations. Six comatose patients were enrolled into a prospective, nonrandomized pilot trial. All patients first received a continuous i.v. infusion of dobutamine (10 microgram. kg-1. min-1) paralleled by continuous administration of midazolam (0.4 mg. kg-1. h-1); thereafter, fentanyl was added i.v. (4 microgram. kg-1. h-1). Hemodynamic parameters as well as dobutamine and endogenous catecholamines plasma levels were determined. The mean arterial blood pressure did not change significantly during the whole study period. The continuous administration of dobutamine (steady-state plasma concentrations: 217 +/- 118 ng. ml-1) increased the beta1-adrenergic receptor-mediated hemodynamic parameters such as heart rate, stroke volume index, cardiac index, and oxygen delivery index (p <.05). The concomitant administration of fentanyl decreased the heart rate-dependent hemodynamic parameters (p <.05), suggesting that fentanyl antagonizes the chronotropic effects of dobutamine. In parallel, dobutamine plasma levels increased significantly (275 +/- 165 ng. ml-1; p <.05). Noteworthy, after administration of fentanyl, oxygen delivery and consumption index returned to baseline values. Radioligand binding experiments on rat cardiac ventricular microsomes ruled out a direct interaction of fentanyl with beta-adrenergic receptors and, more importantly, a fentanyl-induced inhibition of beta-adrenergic receptor G protein coupling. Our observations suggest that fentanyl inhibits the frequency-related hemodynamic changes induced by dobutamine. The underlying mechanism is independent of beta-adrenergic receptors, but is powerful enough to abolish the salutary effect of dobutamine on oxygen delivery and consumption.

Analysis of microdialysates from cancer patients by capillary electrophoresis.

Microdialysis (MD) is an innovative clinical technique for measuring interstitial tissue pharmacokinetics and plasma-to-tissue transfer rates of drugs in humans. However, microdialysis requires the availability of specialized analytical techniques. Capillary electrophoresis (CE), which enables concentration measurements of small volume samples, theoretically constitutes an ideal analytical technique for measuring drug concentrations in microdialysates. In the present experiments, we aimed at assessing the potential utility and limitations of CE for analysis of microdialysates in a clinical situation. Microdialysates were obtained from primary breast cancer patients who received chemotherapy including 5-fluorouracil (5-FU; 600 mg/m2). Subsequently, 5-FU concentrations were measured in tumor - and subcutaneous adipose tissue - microdialysates by CE. By combining MD and CE, complete time versus concentrations profiles could be obtained for 5-FU in the interstitial tumor space and important clinical questions could be addressed. We conclude that the combination of MD and CE leads to important and previously inaccessible information about the drug distribution process in a clinical setting.

Transcription and activity of 5-fluorouracil converting enzymes in fluoropyrimidine resistance in colon cancer in vitro.

Cellular resistance to 5-fluorouracil (5-FU) is not completely understood. Since 5-FU shares the pyrimidine pathway with the physiological pyrimidines, we investigated the relationship between fluoropyrimidine metabolism, nucleic acid uptake and cytotoxicity of 5-FU in eight colon tumour cell lines including 5-FU-resistant subclones. The cytotoxicity of 5-FU was increased up to 423-fold when the anabolites 5-fluorouridine (FUrd), 5-fluorodeoxyuridine (FdUrd), and 5-fluorodeoxyuridine monophosphate (FdUMP) were compared with the parent drug in vitro. The enzymes uridine phosphorylase and thymidine phosphorylase were predictive for the cytotoxicity of 5-FU in 5/7 cell lines. Inhibition of uridine phosphorylase and thymidine phosphorylase by antisense strategies effectively antagonised 5-FU, abolishing 84% and 79% of its toxicity. The importance of thymidine phosphorylase was supported by a highly restricted enzyme activity in 5-FU-resistant cells. In 5-FU naive cells, a stimulating effect of 5-FU on thymidylate synthase mRNA and ribonucleotide reductase mRNA expression was observed. In these cells, antisense oligonucleotides to ribonucleotide reductase significantly reduced cell growth. Downregulation of ribonucleotide reductase mRNA in 5-FU-resistant subclones suggests different mechanisms in primary and secondary resistance to 5-FU. Most of the intracellular 5-FU was selectively incorporated into RNA (range: 45-91%) and generally spared DNA (range: 0.2-11%). In synthesising our data, we conclude that drug resistance could be overwhelmed through bypassing limiting steps in the activation of 5-FU. In the majority of colonic tumours, the activity of uridine phosphorylase and thymidine phosphorylase may have prognostic relevance for the cytotoxicity of 5-FU in vitro.

Exposure of oncologic nurses to methotrexate in the treatment of osteosarcoma.

Methotrexate is a therapeutic agent used widely for osteosarcoma. We used an extremely sensitive high-performance liquid-chromatography assay to evaluate 112 urine samples obtained from 28 hospital employees during high-dose therapy with methotrexate and during routine care of patients. The highest cumulative urinary excretion was observed when methotrexate infusions were handled in a workbench from which a portion of filtered air was emitted into the room. Remarkable urine contaminations were identified for personnel, including 1 administrative employee who had "stood by" for 2 h in the room where infusions were prepared. Lower methotrexate concentrations were detected in the urine of nurses whose exclusive function was to care for patients. The urine burden in oncologic nurses decreased after a central pharmacy unit was installed. Methotrexate was excreted in the sweat of patients who were under high-dose therapy, and its elimination half-life was 11.1 h (mean maximal concentration = 1.7 micrograms/ml [n = 51). The maximal burden in spontaneous vomit from these patients was 441.5 micrograms/ml, and it declined to 0.24 micrograms/ml 19.5 h after infusion was completed. No methotrexate was detected in personnel who prepared 20-g methotrexate infusions in the central pharmacy unit. We demonstrated that occupational safety depended not only on technical precautions, but on the skills of specifically trained personnel.

Stereospecific pharmacokinetics of rac-5-methyltetrahydrofolic acid in patients with advanced colorectal cancer.

1. The pharmacokinetics and toxicity of racemic 5-methyltetrahydrofolic (rac-5-MTHF) acid after i.v. infusion were investigated in 18 patients with advanced colorectal cancer. Doses of 100-600 mg rac-5-MTHF/m2 were administered over 2 h together with a bolus of 500 mg/m2 5-fluorouracil (5-FU) as a midpoint injection. 2. The pharmacokinetics of both diastereoisomers were linear in the range from 100-600 mg 5-MTFH/m2. Independent of the administered dose, the maximal plasma concentration of [R]-5-MTHF was nearly twice that of [S]-5-MTHF. The elimination of [S]-5-MTHF from plasma was considerably faster than that of the [R]-isomer (elimination half-life: 3.1 +/- 1.0 h vs 8.3 +/- 3.2 h). No metabolites were detected in plasma and in urine samples. 3. The plasma protein binding was stereoselective ([R]-5-MTHF bound: 88.2 +/- 2.7%; [S]-5-MTHF bound: 59.9 +/- 6.8%; P < 0.001), causing a significantly higher renal clearance for [S]-5-MTHF when compared with the [R]-isomer (37.5 +/- 23.7 ml min-1 vs 12.7 +/- 11.2 ml min-1, P < 0.001). There was no dose dependence, but gender influenced renal clearance (CLren[R]-5-MTHF: male vs female: 20.5 +/- 14.5 ml min-1 vs 7.8 +/- 4.7 min-1, P = 0.03; CLren [S]-5-MTHF: male vs female: 57.2 +/- 21.7 ml min-1 vs 25.7 +/- 16.2 ml min-1, P = 0.006). 4. Toxic side effects of the combination 5-FU/5-MTHF were rare and generally mild, and included stomatitis, nausea/emesis, diarrhoea, anaemia, leukopenia, and thrombocytopenia. 5. In combination with 500 mg 5-FU/m2 a single dose of 600 mg rac-5-MTHF/m2 can safely be administered to patients with colorectal cancer. A similar therapeutic benefit of 5-MTHF to folinic acid in the biochemical modulation of 5-FU is supported by the comparison of in vitro and in vivo data.

Different stereospecific protein binding of tetrahydrofolates to human serum albumin.

The protein binding of the tetrahydrofolates folinic acid (FA) and its metabolite 5-methyltetrahydrofolic acid (5-MTHF) to human serum albumin (HSA) is stereoselective. At therapeutically relevant concentrations of tetrahydrofolate (range, 5-100 microM), the protein binding was stereoselective to the (R)-isomers of FA and 5-MTHF. The binding of (R)-FA and (R)-5-MTHF was saturated at a concentration of 7% HSA [(R)-tetrahydrofolate bound, ca. 80%]. In contrast to (S)-FA, which was not bound to HSA, (S)-5-MTHF was bound to 45% under physiological conditions. (R)-FA did not influence the protein binding of (S)-FA. Hypoalbuminemia is common in patients with advanced colorectal cancer and affects differentially the protein binding of the diastereoisomers of FA and 5-MTHF. Thus, an influence on the biochemical modulation of 5-fluorouracil by tetrahydrofolates should be taken into consideration.

Pharmacokinetics of rac-leucovorin vs [S]-leucovorin in patients with advanced gastrointestinal cancer.

1. The pharmacokinetics of [R]-leucovorin ([R]-LV), [S]-leucovorin ([S]-LV) and the circulating metabolite [S]-5-methyltetrahydrofolate ([S]-5-MTHF) were studied after administration of racemic LV and [S]-LV in 21 subjects. 2. After intravenous infusion of 600 mg m-2 rac-LV (group 1, n = 7) or 300 mg m-2 [S]-LV (group 3, n = 7), the decay of [S]-LV in plasma was biexponential with a distribution half-life of 0.8 to 1 h and an elimination half-life of 11 to 23 h. When rac-LV was administered as a 2 h i.v. infusion (400 mg m-2) following a loading dose of 200 mg m-2 (group 2, n = 7), the plasma concentrations of [R]-LV and [S]-5-MTHF decayed monoexponentially with mean (+/- s.d.) half-lives of 10 +/- 3 h and 7 +/- 2 h, respectively. 3. The AUC of [S]-5-MTHF was significantly higher after infusion of 300 mg m-2 [S]-LV than after infusion of 600 mg m-2 rac-LV (83 +/- 22 micrograms ml-1 h vs 53 +/- 22 micrograms ml-1 h; P = 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of methotrexate in human urine at nanomolar levels by high-performance liquid chromatography with column switching.

An high-performance liquid chromatographic method with column switching for the detection of less than 4 ng of methotrexate in the urine of oncologic nurses is described. Urine samples were purified by solid-phase extraction on silica-bonded phenyl columns, eluting impurities with ethyl acetate. After elution from the column, the analyte was concentrated ten-fold, evaporating the solvent. On a strong anion-exchange column (Nucleosil 100 SB), methotrexate was separated from the remaining interfering substances, was then switched to a reversed-phase column (LiChrospher 100 RP-18e), and finally eluted by a linear gradient in a solvent system consisting of ammonium formate buffer (pH 2.7) and acetonitrile. Absorbance was monitored at 310 nm. This method has proved to be suitable for detecting traces of methotrexate in urine in order to individualize risks and to reduce further the occupational safety hazard for hospital personnel.