Implementation of point-of-care testing and a temporary influenza ward in a Dutch hospital.

BACKGROUND: The seasonal influenza epidemic poses a significant burden on hospitals, both in terms of capacity and costs. Beds that are occupied by isolated influenza patients result in hospitals temporary being closed to admissions and elective operations being cancelled. Improving hospital and emergency department (ED) patient flow during the influenza season could solve these problems. Microbiological point-of-care-testing (POCT) could reduce unnecessary patient isolation by providing a positive/negative result before admission, but has not yet broadly been implemented. METHODS: A clinical pathway for patients with acute respiratory tract infection presenting at the ED was implemented, including a PCR-based POCT for influenza, operated by nurses and receptionists. In parallel, a temporary ward equipped with 15 beds for influenza-positive patients was established. In this retrospective observational study, we describe the results of implementing this pathway by comparison with the previous epidemic. RESULTS: Clinical performance of the POCT within the clinical pathway was good with strongly decreased time from ED presentation to sample collection (194 vs 47 min) and time from sample collection to result (1094 vs 62 min). Hospital patient flow was improved by a decreased percentage of admitted influenza-positive patients (91% vs 73%) and shorter length of subsequent stay (median 5.86 vs 4.61 days) compared to the previous influenza epidemic. In addition, 430 patient-days of unnecessary isolation have been prevented within a time span of 18 weeks. Roughly estimated savings were almost 400,000 euros. CONCLUSION: We recommend that hospitals explore possibilities for improving patient flow during an influenza epidemic.

Quantitative determination of factors contributing to doxorubicin resistance in multidrug-resistant cells.

There is a large discrepancy between the changes in drug accumulation and the changes in drug cytotoxicity that accompany development of anthracycline resistance in multidrug-resistant cells. In our study, a quantitative relationship has been established between reversal of multidrug resistance by resistance modifiers and a concomitant decrease in intracellular levels of doxorubicin measured at equitoxic concentrations (IC50) in CHRC5 and 2780AD multidrug-resistant cells. (IC50 = concentration required for 50% growth inhibition.) We have demonstrated that resistance modifiers like verapamil and Ro 11-2933/001 act by increasing the effectiveness of intracellular doxorubicin, apparently by inducing redistribution of the drug from the cytoplasm to the nucleus of a multidrug-resistant cell, as shown by quantitative fluorescence microscopy. At complete reversal of resistance, as measured directly or inferred by extrapolation, the amount of intracellular doxorubicin at the IC50 as well as the ratio of nuclear doxorubicin to cytoplasmic doxorubicin were the same as those in sensitive cells. These results offer an explanation for the frequently observed discrepancies between drug accumulation and cytotoxicity and also show quantitatively that a decrease in drug accumulation and a change in intracellular drug distribution together are the only determinants of doxorubicin resistance in the multidrug-resistant cells studied.

Sensitivity of human, murine, and rat cells to 5-fluorouracil and 5'-deoxy-5-fluorouridine in relation to drug-metabolizing enzymes.

Six cell lines differing in histological origin were studied regarding the growth inhibitory effect of fluoropyrimidines in relation to their metabolism. The human colon carcinoma cell line WiDr was most sensitive to 5-fluorouracil (FUra) (50% growth inhibitory concentration, 0.7 microM) and to its analogue 5'deoxy-5-fluorouridine (5'dFUR) (50% growth inhibitory concentration, 18 microM). The murine B16 melanoma cell line was moderately sensitive to FUra but least sensitive to 5'dFUR. The 50% growth inhibitory concentration values in the human melanoma cell lines IGR3 and M5, the transformed human intestine cell line intestine 407 and the rat hepatoma cell line H35 varied for FUra between 1.7 and 5.0 microM, and for 5'dFUR between 54 and 160 microM. Several enzymes from pyrimidine metabolism responsible for FUra metabolism were measured with FUra as a substrate. The activity of uridine phosphorylase, which catalyzes the conversion of 5'dFUR to FUra, was lowest in B16 cells correlating with the low sensitivity to 5'dFUR. When adenosine 5'-triphosphate was included in the reaction mixture for uridine phosphorylase, FUra was rapidly channeled into FUra nucleotides via its nucleoside. The rate of channeling appeared to correlate with the nucleoside phosphorylase activity in the various cell lines. In several cell lines activities of nucleotide-degrading enzymes were rather high and interfered with the measurement of orotate phosphoribosyl transferase (OPRT) with FUra as substrate. Addition of the phosphatase inhibitor glycerol-2-phosphate partly prevented breakdown of the newly formed 5-fluorouridine 5'-monophosphate and enabled measurement of OPRT. The WiDr cell line had a relatively high OPRT activity which could explain its sensitivity to FUra. The activity of thymidylate synthase was measured at a suboptimal concentration of 1 microM and at the optimal concentration of 10 microM deoxyuridine 5'-phosphate. With all cell lines the ratio between the activities at 10 and 1 microM was between 2.3 and 3.6. The activity of thymidylate synthase was lowest in WiDr and IGR3 cells and 3-4 times higher in M5 and Intestine 407 cells. The inhibition of 0.01 microM 5-fluorodeoxyuridine 5'-monophosphate was 80-90% at 1 microM deoxyuridine 5'-phosphate and 50-70% at 10 microM deoxyuridine 5'-phosphate with all cell lines. At 0.1 microM 5-fluorodeoxyuridine 5'-monophosphate enzyme activity was inhibited by 95-100%. The incorporation of FUra into RNA was relatively low in IGR3 cells and 3-5 times higher in all other cell lines.(ABSTRACT TRUNCATED AT 400 WORDS)

Energy-dependent processes involved in reduced drug accumulation in multidrug-resistant human lung cancer cell lines without P-glycoprotein expression.

Mechanisms contributing to reduced cytotoxic drug accumulation were studied in two multidrug-resistant (MDR) human lung cancer cell lines without P-glycoprotein expression. In these (non-small cell) SW-1573/2R120 and (small cell) GLC4/ADR MDR cells, the steady-state accumulation of [14C]daunorubicin was 30 and 12%, respectively, of that in the parent cells. When cells, at steady state, were permeabilized with digitonin, the amount of daunorubicin binding increased only in the resistant cells. The reduced accumulation of daunorubicin in the SW-1573/2R120 and GLC4/ADR cells was accompanied by a lower initial (2 min) uptake rate of this drug. No difference in initial efflux rate of daunorubicin from preloaded cells could be detected between sensitive and resistant SW-1573 cells. However, daunorubicin was extruded 5-fold faster from GLC4/ADR cells than from the parental cells. In the presence of the energy metabolism inhibitors sodium azide and deoxyglucose, the reduced daunorubicin accumulations in the SW-1573/2R120 and GLC4/ADR MDR cells were (almost) completely reversed. The effects of these inhibitors on drug uptake were already apparent during the earliest measured time points (less than 15 s). Also, the enhanced efflux of daunorubicin from GLC4/ADR cells was inhibited. In ATP-depleted cells, the intracellular pH was lowered by approximately 0.3 units in resistant as well as in sensitive cells. The lower intracellular pH, however, could not account for the increase in daunorubicin accumulation in the resistant cells. Also, for vincristine and etoposide, the increases in drug accumulation under energy-deprived conditions were more pronounced in the resistant SW-1573/2R120 cells than in the parent SW-1573 cells. These results suggest that accumulation of drugs in the non-P-glycoprotein MDR human lung carcinoma cell lines SW-1573/2R120 and GLC4/ADR is reduced by an energy-dependent drug export mechanism which prevents efficient transport of drug to the target. Since P-glycoprotein expression in lung tumors is generally low, these MDR lung cancer cell lines can be used as a model to study alternative mechanisms leading to multidrug resistance in this tumor type.

Pharmacokinetics of 5-fluorouracil assessed with a sensitive mass spectrometric method in patients on a dose escalation schedule.

The pharmacokinetics of 5-fluorouracil (5-FUra) was investigated in 21 patients with advanced cancer (mainly colorectal cancer). 5-FUra was administered as weekly i.v. bolus injections usually at a starting dose of 500 mg/m2. Every 4 weeks the dose was escalated by 20% until dose-limiting toxicity was observed. Whenever possible, pharmacokinetic studies were performed at dose escalation. 5-FUra plasma concentrations were measured by high pressure liquid chromatography and a sensitive gas chromatography-mass spectrometry assay with a sensitivity as low as 3 x 10(-9) M. According to the 42 plasma concentration versus time curves, in all but one of the patients total body clearance decreased with increasing 5-FUra doses, consistent with the nonlinear pharmacokinetics of 5-FUra. The ultrasensitive assay revealed an almost horizontal phase in the plasma concentration versus time curves at plasma concentrations of 10(-8) to 10(-9) M. This plateau did not show correlation with the area under those curves. The use of a logistic regression method showed that clinical toxicity was correlated with the area under the plasma concentration versus time curve of 5-FUra.

Correlation of multidrug resistance with decreased drug accumulation, altered subcellular drug distribution, and increased P-glycoprotein expression in cultured SW-1573 human lung tumor cells.

Four multidrug-resistant variants of the human squamous lung cancer cell line SW-1573 with levels of doxorubicin resistance ranging from 10- to 2000-fold were characterized with respect to drug accumulation and efflux, subcellular drug distribution pattern, antioxidant defenses, and P-glycoprotein expression. For all these parameters except the antioxidant defenses a correlation was observed with the level of doxorubicin resistance; with increasing drug resistance cellular drug accumulation capacity (as measured for doxorubicin) progressively decreased, initial drug efflux rates (as measured for daunorubicin) progressively increased, while the subcellular doxorubicin distribution (as measured by fluorescence microscopy) gradually shifted from a "mainly nuclear" to a "mainly cytoplasmic" pattern. Our data suggest that in the present set of cell lines the same mechanism of resistance is operating at all levels of doxorubicin resistance.

Phase I and pharmacokinetic studies of high-dose uridine intended for rescue from 5-fluorouracil toxicity.

The clinical effects and pharmacokinetics of high-dose uridine were determined in seven patients with advanced-stage cancer and in one healthy volunteer. Uridine was also examined for its effect on 5-fluorouracil toxicity in two patients. Uridine was administered as a 1-hr i.v. infusion at doses of 1 to 12 g/sq m. Plasma and urine samples were analyzed for uridine and uracil using high-pressure liquid chromatography. In 23 courses of uridine alone, the only toxicity observed was transient shivering after one of two courses at 12 g/sq m. This side effect was also seen after administration of uridine (10 g/sq m) during combination with 5-fluorouracil. The pretreatment plasma uridine concentration was elevated from low micromolar to millimolar levels with uridine administration at doses up to 12 g/sq m. Maximal areas under the concentration-time curve were about 5 mmol/liter/hr. Both peak plasma level and area under the curve for uridine increased linearly with dose. Uridine plasma decay curves were biphasic with a terminal half-life of 118 min. Half-life, volume of distribution (634 ml/kg), and total clearance (4.98 ml/kg/min) appeared to be independent of dose. Plasma uracil concentration increased gradually after administration of uridine to plateau levels. Maximal plasma uracil concentrations were about one-tenth that of peak uridine concentrations. The plasma uracil level declined with a half-life of about 40 min after uridine levels decreased to 300 microM. Total urinary excretion of uridine was 24% of the dose, while the amount of uracil recovered in urine was 3.4%. In two patients, uridine rescue was attempted during 5-fluorouracil dose escalation. Uridine at 5 to 6 g/sq m given on 1 or on 2 days after 5-fluorouracil did not prevent myelosuppression and gastrointestinal toxicity associated with increasing plasma concentrations of 5-fluorouracil. These data show that uridine administered as a 1-hr infusion at doses which provide peak plasma uridine concentrations in the millimolar range is well tolerated. Rapid elimination of uridine primarily due to catabolism results in modest exposure to substantially elevated plasma uridine concentrations. Preliminary findings suggest that prolonged treatment with uridine may be required to test its potential to rescue patients from 5-fluorouracil toxicity.

Saturable P-glycoprotein kinetics assayed by fluorescence studies of drug efflux from suspended human KB8-5 cells.

This article describes a new and rapid method to determine the pumping rate of P-glycoprotein (P-gp) in intact cells. Multidrug resistant (MDR) human epidermoid carcinoma KB8-5 cells (containing P-gp) were loaded with daunorubicin (DNR) in the absence or in the presence of verapamil, sufficient to inhibit DNR pumping by P-gp. In either case, the cells were resuspended in medium devoid of DNR and the subsequent increase of the DNR fluorescence intensity was measured as a function of time. For cells loaded with the same amount of drug, the free cytosolic drug concentration (Ci(t)) was a unique function of the DNR medium concentration (Co(t)). The cellular drug content in the presence of verapamil decreased nonlinearly with decreasing extracellular drug concentration, indicating that the intracellular drug apparent distribution volume increased with decreasing cellular drug content. At each fluorescence intensity, we calculated the P-gp mediated (verapamil-inhibitable) DNR transport rate from the rate of increase of the DNR fluorescence intensity in the absence of verapamil minus the rate of increase of the DNR fluorescence intensity in the presence of verapamil. When plotted against the intracellular free drug concentration (as calculated from the total cellular drug content and a separately determined relation between the total cellular drug content and the intracellular free drug concentration: the apparent distribution volume), this P-gp mediated DNR transport rate showed saturation of P-gp at higher DNR concentrations. The results imply that P-gp mediated DNR transport is saturable (the value of Km is in the order of 1 microM).

Evidence for daunomycin efflux from multidrug-resistant 2780AD human ovarian carcinoma cells against a concentration gradient.

Using a flow-through system human multidrug-resistant 2780AD ovarian carcinoma cells were exposed to flowing culture medium containing the anticancer agent daunomycin (5 microM). A pulse of medium containing verapamil caused increased cellular daunomycin accumulation, resulting in a dip in the fluorescence signal from daunomycin in the effluent. After passage of this pulse we observed an efflux of more than 90% of this extra accumulated daunomycin within 10 min. This daunomycin efflux against a concentration gradient provides evidence for drug efflux from these cells being an active process. After the addition of methylamine to the medium to increase intravesicular pH, the dip in the fluorescence signal was not decreased, indicating that vesicular transport was not an important component of this efflux.

In-beam light absorption measurement of anthracyclines in cells with a flow-through system.

A flow-through cuvette in which cells attach as a monolayer to a quartz plate was developed for measurement of the light absorbance of anthracyclines in cells. Despite the drawback of a short path-length (of the order of the cell diameter), a dynamic flow-through set-up and baseline storage made it possible to measure intracellular absorbance and obtain spectral data for daunomycin and carminomycin. Stopping the flow and allowing the drug to equilibrate between medium and cells led to a 20% decrease of molar light absorption of cellular anthracycline, which permitted measurement of the total cellular concentration. Furthermore, accumulation and efflux kinetics were determined for H35 rat hepatoma cells. On the basis of the reported formation constant of the iron-complex of carminomycin, which is of the order of 10(34), we expected to find this complex within the cells. However, the spectrum of cellular drug did not show absorbance bands characteristic of the complex. A red shift and hypochromism were found in the daunomycin spectrum after intracellular binding, which corresponds with the spectral change observed after intercalation of daunomycin into DNA.

Anthracyclines modulate multidrug resistance protein (MRP) mediated organic anion transport.

We studied the ATP-dependent uptake of dinitrophenyl-glutathione (GS-DNP) into plasma membrane vesicles derived from parental GLC4 cells and from multidrug resistant GLC4/ADR cells. The latter have a high expression of the multidrug resistance protein (MRP). Uptake of GS-DNP into membrane vesicles from GLC4/ADR cells was highly stimulated by the addition of ATP, compared to the uptake into membrane vesicles from GLC4 cells. This ATP-dependent uptake into membrane vesicles from GLC4/ADR cells was saturable with a Km of 1.2 +/- 0.2 microM and a Vmax of 560 +/- 80 pmol/mg prot./min. ATP stimulated GS-DNP uptake with a Km of 187 +/- 4 microM. This uptake was specifically inhibited by a polyclonal serum raised against a fusion protein containing a segment of MRP. The ATP-dependent uptake of GS-DNP was not only inhibited by organic anions, such as oxidized glutathione (GSSG), methotrexate (MTX) and some bile acids, but also by non-anionic natural product drugs, such as anthracyclines, vinca alkaloids and etoposide (VP-16). Uptake of GSSG and MTX into membrane vesicles from GLC4/ADR cells could be stimulated by ATP. The ATP-dependent uptake of GSSG had a Km of 43 +/- 3 microM and a Vmax of 900 +/- 200 nmol/mg protein/min. The ATP-dependent uptake of GS-DNP seemed to be non-competitively inhibited by the anthracycline daunorubicin (DNR), whereas the ATP-dependent GSSG uptake seemed to be competitively inhibited by DNR. A substrate binding site on MRP is proposed that comprises a pocket in which both DNR and GS-DNP or GSSG bind in random order to different, only partly overlapping sites. In this pocket binding of a second compound is influenced by the compound which was bound first.

Cellular daunomycin fluorescence in multidrug resistant 2780AD cells and its relation to cellular drug localisation.

Multidrug resistant (MDR) 2780AD human ovarian carcinoma cells were loaded with the fluorescent anticancer agent daunomycin (DN). Fluorescence anisotropy was lower than for corresponding A2780 wild-type cells, indicating that DN was less rigidly bound than in the wild-type cells. Average fluorescence quenching of DN was lower for 2780AD cells. Data were fitted into a model with a highly quenched fraction (fraction A), corresponding to DN intercalated in DNA, and an unquenched fraction (fraction B). The ratio A/B was one order of magnitude lower for the MDR cells than for the wild-type cells. Two other MDR cell lines were investigated and low A/B ratios were found in both cases. Thus, evidence has been provided that in MDR cells the DNA-bound fraction is relatively low and that more free DN is present, for example in acidic vesicles.

Simvastatin improves disturbed endothelial barrier function.

BACKGROUND: Recent clinical trials have established that inhibitors of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) reduce the risk of acute coronary events. These effects of statins cannot be fully explained by their lipid-lowering potential. Improved endothelial function may contribute to the positive effects of statin treatment. METHODS AND RESULTS: In the present study, we report that simvastatin reduces endothelial barrier dysfunction, which is associated with the development of atherosclerosis. Treatment of human umbilical vein endothelial cells for 24 hours with 5 micromol/L simvastatin reduced the thrombin-induced endothelial barrier dysfunction in vitro by 55+/-3%, as assessed by the passage of peroxidase through human umbilical vein endothelial cell monolayers. Similar effects were found on the thrombin-induced passage of (125)I-LDL through human aortic endothelial cell monolayers. This reduction in barrier dysfunction by simvastatin was both dose and time dependent and was accompanied by a reduction in the thrombin-induced formation of stress fibers and focal adhesions and membrane association of RhoA. Simvastatin treatment had no effect on intracellular cAMP levels. In Watanabe heritable hyperlipidemic rabbits, treatment for 1 month with 15 mg/kg simvastatin reduced vascular leakage in both the thoracic and abdominal part of the aorta, as evidenced by the Evans blue dye exclusion test. The decreased permeability was not accompanied by a reduction of oil red O-stainable atherosclerotic lesions. CONCLUSIONS: These data show that simvastatin, in a relatively high concentration, improves disturbed endothelial barrier function both in vitro and in vivo. The data also support the beneficial effects of simvastatin in acute coronary events by mechanisms other than its lipid-lowering effect.

P-glycoprotein in primary acute myeloid leukemia and treatment outcome of idarubicin/cytosine arabinoside-based induction therapy.

The expression of the drug transport protein, P-glycoprotein (Pgp/MDR1) has been found to be of prognostic significance for the achievement of complete remission (CR) or the duration of survival after daunorubicin (DNR)-containing induction therapy in acute myeloid leukemia (AML). This would suggest that the expression of Pgp in AML is high enough to have significant impact on intracellular DNR concentrations and on clinical therapy failure in AML. Recently, DNR has been replaced in many centers by idarubicin (IDA) as the first choice anthracycline in AML treatment. We have, therefore, performed a study in a group of 98 primary AML patients, who all received IDA, but not DNR during induction therapy in order to determine if the response to IDA-containing induction therapy might be related to the biologic characteristic of Pgp expression in AML. The AML samples were studied for Pgp expression by MRK16 antibody staining and for Pgp activity measured as the modulation of rhodamine 123 uptake by 2 microM PSC 833. No correlation of Pgp with complete response rate, event-free survival or overall survival was found. In addition to Pgp, the expression of another protein that has been implicated by some studies in response failure to DNR-containing therapy, the major vault protein (Mvp/LRP), was studied. This marker did not correlate with CR or survival after IDA-containing therapy. The results of this patient study are consistent with model studies showing that the steady-state cellular accumulation of lipophilic anthracyclines such as IDA are little affected by Pgp. Therefore, putative beneficial effects of the inclusion of PSC 833 in IDA-containing therapy might rather be related to alternative mechanisms than to inhibition of Pgp-mediated IDA efflux.

Theoretical and practical considerations for the measurement of P-glycoprotein function in acute myeloid leukemia.

This paper summarizes experimental data and theoretical considerations, that are important for the measurement of P-glycoprotein (Pgp) function in acute myeloid leukemia (AML). The data are presented in subdivisions based on the techniques used, which will facilitate finding specific information. Based on our extensive experience with Pgp analysis, which includes radioactive assays, flow cytometry and fluorescence microscopy, we recommend a flow cytometry-based assay, that measures the effect of 2 microM PSC 833 on rhodamine 123 (R123) accumulation as the most practical and sensitive functional Pgp test. In combination with the flow cytometric measurement of Pgp using an antibody against an extracellular epitope (eg MRK16), this offers a sensitive and reproducible method for Pgp detection in AML, which is also rapid and practical. Furthermore, an R123 accumulation assay is specific for Pgp, because R123 is transported much less efficiently by the multidrug resistance protein (MRP) than by Pgp. Another probe of similar sensitivity and specificity is 3,3'-diethyloxacarbocyanine iodide. Alternatively, especially for the analysis of small numbers of cells (for example sorted subpopulations of leukemic cells), convenient and sensitive procedures are being developed by using DNA-binding Pgp substrates which remain fixed in the nuclei of the cells upon formaldehyde exposure for quantitative fluorescence laser scanning microscopy with image analysis. Less experimental data have been published to establish the optimal conditions for dual parameter flow cytometry (Pgp function, in eg Pgp+ or CD34+ cells). However, laboratories with flow cytometry experience will be able to implement this useful option to analyze subpopulations of cells.

Do P-glycoprotein and major vault protein (MVP/LRP) expression correlate with in vitro daunorubicin resistance in acute myeloid leukemia?

Two proteins that have been correlated with the occurrence of multidrug resistance in acute myeloid leukemia (AML) are P-glycoprotein (Pgp) and the major vault protein (Mvp/LRP). With the purpose of further quantifying the potential contributions of Pgp-mediated drug efflux and Mvp/LRP to drug resistance in AML we have investigated whether the transport function of Pgp and the expression of Mvp/LRP correlated with the accumulation of daunorubicin (DNR) and the in vitro resistance to DNR cytotoxicity (LC50 by MTT assay) in AML cells. In de novo adult AML, the steady-state DNR accumulation (in pmol/10(6) cells) correlated with Pgp activity or expression, whereas the LC50 for DNR did not correlate with Pgp activity (measured as the modulation of rhodamine 123 or DNR accumulation by the Pgp inhibitor PSC833) or Pgp expression (measured by flow cytometry with the MRK-16 antibody). The contribution of MRP1 expression to a reduced DNR accumulation seems minor compared to Pgp. In addition, the modulation of the DNR LC50 by PSC833 did not correlate with Pgp protein or activity. The steady-state DNR accumulation showed no correlation with the DNR LC50. The Mvp/LRP expression (immunocytochemical staining) did neither correlate with DNR accumulation nor with the DNR LC50. A significant negative correlation was seen between the Mvp/LRP immunocytochemical staining and Pgp activity, indicating that both markers define (partially) different populations. In conclusion, it is shown that Pgp function, but not Mvp/LRP or MRP1 expression correlate with a low steady-state DNR accumulation in de novo AML. The Pgp activity does, however, not predict the DNR sensitivity in AML measured as in vitro DNR LC50 with an MTT-based assay. The reason for that seems to be that a low DNR accumulation may not be the most important factor in determining the LC50. While the clinical usefulness of these drug resistance tests remains to be proven they do not seem to provide as yet a straightforward explanation for the major cause(s) of clinical chemotherapy failure.

Doxorubicin gradients in human breast cancer.

Ten patients with locally advanced breast cancer were given doxorubicin i.v., and an incision biopsy was subsequently taken. Doxorubicin autofluorescence was examined using computerized laser scanning microscopy, and microvessels were immunostained in the same sections. Overlays of both pictures revealed doxorubicin gradients in tumor islets with high concentrations in the periphery and low concentrations in the center of the tumor islets. Gradients were most pronounced shortly after the injection, but they could still be detected 24 h later. No gradients were observed in connective tissue. This study demonstrates a serious risk of the drug not reaching all of the cancer cells in those cases in which the cancer cells are densely packed in islets. The efficacy of drug treatment will thus depend on the histology of the tumor tissue.

Pharmacokinetics of bolus 5-fluorouracil: relationship between dose, plasma concentrations, area-under-the-curve and toxicity.

The pharmacokinetics of 5-fluorouracil (5FU) have been related to toxicity and antitumor activity, in particular for continuous infusion schedules, but to a lesser extent for frequently used bolus injections. The use of intensive sampling schedules limits the application of pharmacokinetics to optimize individual dosing or to define the ideal combination with other drugs. We therefore reanalyzed a pharmacokinetic study in order to develop a limited sampling schedule. Patients received escalating doses of 5FU at 500, 600 and 720 mg/m2 as a bolus until toxicity developed. Blood samples were analyzed until 24 h after administration. The area under the concentration time curve from 0-90 min (AUC(0-90)) was strongly correlated with dose and also with toxicity (p = 0.0009). The 5FU concentrations at 30 and 60 min were correlated to the AUC(30-240) and to that of the AUC(0-90) (r2 = 0.970). The use of limited sampling (30, 60, 90 min) in a patient given 353 mg/m2 5FU with severe toxicity at initial dosing at 500 mg/m2 revealed that the AUC(0-90) at 353 mg/m2 was higher than the normal AUC(0-90) for 500 mg/m2. This patient appeared to have an 8-fold lower activity of the 5FU degradation enzyme dihydropyrimidine dehydrogenase. Limited sampling will allow us to define potential aberrant kinetics of pharmacokinetic interaction of 5FU with other drugs being developed for treatment of colorectal cancer.

The gut microbiota in internal medicine: implications for health and disease.

The human gut microbiota may be viewed as an organ, executing numerous functions in metabolism, development of the immune system and host defence against pathogens. It may therefore be involved in the development of a range of diseases such as gastrointestinal infections, inflammatory bowel disease, allergy and diabetes mellitus. Reversely, certain therapies that are often used, such as antibiotics and chemotherapy, may negatively affect the composition and function of the gut microbiota and thereby the wellbeing of patients. As the microbiota research field is currently moving from association studies to intervention studies and even clinical trials, implementation of this new knowledge into clinical practice is coming near. Several therapeutic interventions that target the gut microbiota are being evaluated, ranging from supplementation of food components to transplantation of faecal microbiota. In this review we provide an overview of current literature on the gut microbiota in both a healthy state and a range of diseases that are relevant for internal medicine. In anticipation of gut microbiota-targeted therapies, it is important to realise the key function of the gut microbiota in physiological processes and the collateral damage that may be caused when disrupting this ecosystem within us.

Cooperative P-glycoprotein mediated daunorubicin transport into DNA-loaded plasma membrane vesicles.

Most of the multidrug resistant human tumor cell lines overexpress the MDR1 gene product P-glycoprotein (P-gp) which is believed to function as an energy-dependent drug efflux pump. Here we describe a novel method that allows the kinetic characterization of P-gp-mediated active drug transport. This method is based on the fluorescence quenching of anthracyclines transported into DNA-loaded plasma membrane vesicles. The uptake of daunorubicin (DNR) into the plasma membrane vesicles was saturable in terms of the extravesicular DNR concentration with a Km of 1.5 +/- 0.1 microM. This transport occurred by a cooperative process with a Hill coefficient close to 2 for DNR. A model is discussed in which P-gp pumps two molecules of drug per turnover.