Measurement of in vitro cellular pharmacokinetics of 5-fluorouracil in human and rat cancer cell lines and rat hepatocytes using a flow-through system

Summary A flow-throught system was used to study the cellular pharmacokinetics of 5-fluorouracil (5-FU) in four human cell lines (squamous-cell carcinoma HEp-2, colon carcinoma WiDr, hepatoma Hep G2, and breast carcinoma MCF-7) as well as in the rat hepatoma H35 cell line and in freshly isolated rat hepatocytes. The system made it possible to restrict the decrease in the concentration of 5-FU in the medium, to keep the volume in which the metabolites accumulated relatively small, and to study the dynamics of a response during and after a change in the composition of the eluent. Clearance of 5-FU from the eluent was achieved predominantly (>95%) by its catabolism to dihydrofluorouracil in the tumor cell lines and to 2-fluoro--alanine in the hepatocytes. Not only rat hepatocytes but also HEp-2 cells showed relatively high clearance values. A concentration-dependent 5-FU elimination was observed, indicating saturation of 5-FU elimination according to Michaelis-Menten kinetics (K_m 14–22 M). The maximal velocity (V_max) values ranged from 0.025 to 0.13 nmol 5-FU/10⁶ cells per minute. For HEp-2 cells, high-concentration pulse injections of 5-FU, thymine, uridine, or uracil immediately led to a reduction in 5-FU conversion, followed by recovery within 5 min. The flow-through system proved to be adequate for the study of the non-linear pharmacokinetics of 5-FU in different intact cells and for the comparison of various manipulations of these pharmacokinetics.Abbreviations 5-FU 5-fluorouracil - FUR 5-fluorouridine - F-DHU dihydrofluorouracil - F--ala 2-fluoro--alanine - F-UPA -fluoroureidopropionic acid - HEPES 4-(2-hydroethyl)-1-piperazine-ethane sulfonic acid - MEM modified minimal essential medium - HBSS Hanks' balanced salt solution - HPLC high-performance liquid chromatography Supported by grant IKA 87-16 from the Netherlands Cancer Foundation. One author (G. J. P.) is the recipient of a senior research fellowship from the Royal Netherlands Academy of Arts and Sciences (KNAW)     

Staged TRAM Breast Reconstruction: Combining the Advantages of Tissue Expansion with Surgical Delay

The TRAM flap has become the gold standard in breast reconstruction but suffers from the disadvantages of poor color match, different texture, and impaired sensation compared to the normal breast. This study reports on a two-stage procedure to address these problems. The first stage consists of insertion of a tissue expander and surgical delay of the TRAM flap. The second stage consists of removal of the tissue expander and transposition of a deepithelized TRAM flap into the tissue expanded cavity. (The capsule is excised.) Four cases of breast reconstruction are reported. The advantage of this procedure is that it offers the benefits of tissue expansion, viz., normal color match, texture, and sensation, and in addition, reconstruction is achieved with autologous tissue by a pedicled TRAM flap. The vascularity of the TRAM is enhanced by a surgical delay procedure.     

Effects of skyrin, a receptor-selective glucagon antagonist, in rat and human hepatocytes

Peptidic glucagon antagonists have been shown to lower blood glucose levels in diabetic models (1-3), but attempts to identify small molecular weight glucagon receptor-binding antagonists have met with little success. Skyrin, a fungal bisanthroquinone, exhibits functional glucagon antagonism by uncoupling the glucagon receptor from adenylate cyclase activation in rat liver membranes (1). We have examined the effects of skyrin on cells transfected with the human glucagon receptor and on isolated rat and human hepatocytes. The skyrin used was isolated from Talaromyces wortmanni American Type Culture Collection 10517. In rat hepatocytes, skyrin (30 micromol/l) inhibited glucagon-stimulated cAMP production (53%) and glucose output (IC50 56 micromol/l). There was no detectable effect on epinephrine or glucagon-like peptide 1 (GLP-1) stimulation of these parameters, which demonstrates skyrin's selective activity. Skyrin was also evaluated in primary cultures of human hepatocytes. Unlike cell lines, which are largely unresponsive to glucagon, primary human hepatocytes exhibited glucagon-dependent cAMP production for 14 days in culture (EC50 10 nmol/l). Skyrin (10 micromol/l) markedly reduced glucagon-stimulated cAMP production (55%) and glycogenolysis (27%) in human hepatocytes. The inhibition of glucagon stimulation was a specific property displayed by skyrin and oxyskyrin but not shared by other bisanthroquinones. Skyrin is the first small molecular weight nonpeptidic agent demonstrated to interfere with the coupling of glucagon to adenylate cyclase independent of binding to the glucagon receptor. The data presented in this study indicate that functional uncoupling of the human glucagon receptor from cAMP production results in metabolic effects that could reduce hepatocyte glucose production and hence alleviate diabetic hyperglycemia.     

Induction of p53 up-regulated modulator of apoptosis messenger RNA by chemotherapeutic treatment of locally advanced breast cancer.

PURPOSE: In biopsies of patients with locally advanced breast cancer, we investigated the in vivo changes of the gene expression pattern induced by chemotherapy to find genes that are potentially responsible for the efficacy of the drug. EXPERIMENTAL DESIGN: Early cellular responses to chemotherapy-induced damage, both in vivo and in vitro, were investigated by analyzing chemotherapy-induced changes in gene expression profiles. Core biopsies were taken from nine patients with locally advanced breast cancer, before and at 6 hours after initiation of doxorubicin-based chemotherapy. Both samples were cohybridized on the same microarray containing 18,000 cDNA spots. RESULTS: The analysis revealed marked differences in gene expression profile between treated and untreated samples. The gene which was most frequently found to be differentially expressed was p53 up-regulated modulator of apoptosis (PUMA). This gene was up-regulated in eight of nine patients with an average factor of 1.80 (range, 1.36-2.73). In vitro MCF-7 breast cancer cells exposed to clinically achievable doxorubicin concentrations for 6 hours revealed marked induction of PUMA mRNA, as well. CONCLUSIONS: This is the first report describing PUMA mRNA to be up-regulated as a response to chemotherapy in patients. Because PUMA is a known member of the family of BH3-only proapoptotic proteins, this finding suggests PUMA's potential importance for the response to anticancer drugs.     

Lack of efficacy of two consecutive treatments of radioimmunotherapy with 131I-cG250 in patients with metastasized clear cell renal cell carcinoma.

PURPOSE: A previous activity dose-escalation study using 131I-labeled chimeric monoclonal antibody cG250 in patients with progressive metastatic renal cell carcinoma (RCC) resulted in occasional therapeutic responses. The present study was designed to determine the safety and therapeutic efficacy of two sequential high-dose treatments with 131I-cG250. PATIENTS AND METHODS: Patients (n = 29) with progressive metastatic RCC received a low dose of (131)I-cG250 for assessment of preferential targeting of metastatic lesions, followed by the first radioimmunotherapy (RIT) with 2220 MBq/m2 131I-cG250 (n = 27) 1 week later. If no grade 4 hematologic toxicity was observed, a second low-dose 131I-cG250 (n = 20) was given 3 months later. When blood clearance was not accelerated, a second RIT of 131I-cG250 was administered at an activity-dose of 1110 MBq/m2 (n = 3) or 1665 MBq/m2 (n = 16). Patients were monitored weekly for toxicity, and tumor size was evaluated by computed tomography once every 3 months intervals. RESULTS: The maximum-tolerated dose (MTD) of the second RIT was 1,665 MBq/m2 because of dose-limiting hematological toxicity. Based on an intention-to-treat analysis, after two RIT treatments, the disease stabilized in five of 29 patients, whereas it remained progressive in 14 of 29 patients. Two patients received no RIT, and eight of 29 received only one 131I-cG250 RIT because of grade 4 hematologic toxicity, formation of human antichimeric antibodies, or disease progression. CONCLUSION: In patients with progressive end-stage RCC, the MTD of the second treatment was 75% of the MTD of the first RIT. In the majority of patients, two cycles of 131I-cG250 could be safely administered without severe toxicity. No objective responses were observed, but occasionally two RIT doses resulted in stabilization of previously progressive disease.     

Uptake of estradiol or progesterone into the CSF following intranasal and intravenous delivery in rats.

The uptake of estradiol and progesterone into the cerebrospinal fluid (CSF) after intranasal and intravenous administration in rats was investigated. Each animal received estradiol intranasally (40 microg/rat) and by intravenous infusion (10 microg/rat) into the jugular vein using a vascular access port. Hereafter, the same set of rats was treated with progesterone intranasally (200 microg/rat) and by intravenous infusion (104 microg/rat). Following nasal delivery, both steroid hormones reach Cmax values in plasma and CSF at 15 min after administration. Intravenous infusion of estradiol and progesterone shows comparable plasma and CSF concentration-time profiles compared to the nasal route. For both hormones the AUCCSF/AUCplasma ratios (mean +/- SD) after intranasal delivery (estradiol 2.3 +/- 1.1%; progesterone 1.9 +/- 0.7%) do not differ significantly from the ratios shown after intravenous infusion (estradiol 2.0 +/- 0.6%; progesterone 2.2 +/- 0.8%). These results indicate that after nasal delivery estradiol and progesterone are rapidly absorbed into the systemic circulation, from where the non-protein bound hormones probably enter the CSF by crossing the blood-brain barrier. No extra direct nose-CSF transport could be demonstrated.     

Costs and effects of secondary prevention with perindopril in stable coronary heart disease in Poland: an analysis of the EUROPA study including 1251 Polish patients

OBJECTIVES: To estimate the long-term impact of treatment with perindopril on costs and health effects in patients with stable coronary artery disease in Poland. METHODS: The cost-effectiveness analysis was based on data from a randomized double-blind, placebo-controlled trial. A decision-tree analysis was employed, including Monte Carlo and bootstrapping techniques. This study was a sub-study of the EUROPA (European Trial on Reduction of Cardiac Events with Perindopril in Stable Coronary Artery Disease) trial (n = 12 218; mean follow-up 4.2 years). Resource use was based on data from Polish EUROPA study patients (n = 1251), while effectiveness was based on the whole EUROPA study. The health gain of perindopril in life-years was based on overall EUROPA study results, and the adapted Polish life expectancy of patients not dying during the trial. Costs were calculated in new Polish zloty (PLN), year 2003 values; euro1 = PLN4.053. Only direct healthcare costs related to cardiovascular events and medication use were studied. RESULTS: When observed mortality was combined with life expectancy beyond the end of the study, perindopril use showed a gain in life expectancy of 0.182 life-years (SD +/- 0.129) at a cost of PLN1983 (SD +/- 103) with discounting of 5% per annum on costs and no discounting on effects. This resulted in an incremental cost-effectiveness ratio (ICER) of PLN10 896 per life-year gained. The probability that the ICER for perindopril was below the threshold of PLN60 000 was 88%. The overall results were insensitive to discount rates for costs and life-years. CONCLUSIONS: Perindopril leads to a reduction in the risk of coronary events among patients with stable heart disease. When the expected improvement in life expectancy is combined with associated medical costs, there is a high probability that perindopril is cost effective, given the threshold of PLN60 000 per life-year gained.