Prediction of thioguanine-induced cytotoxicity by dual-parameter flow cytometric analysis

A method is presented for the quantitative analysis of delayed cytokinetic effects resulting from the treatment of L1210 cells with 6-thioguanine (TG). By using dual-parameter (DNA/protein) flow cytometry, we could observe the accumulation of late S/G2/M cells with abnormally high green fluorescence (i.e., protein content), indicative of unbalanced growth. The use of mitotic cells from a pseudotetraploid line (HT29) as external markers for both red and green fluorescence facilitated highly reproducible measurement of the mean green fluorescence (GFL mean ) of the arrested late S/G2/M population. We found that the dose dependence of the observed GFL mean values followed the same unusual biphasic pattern as did cytotoxicity in this cell line, indicating that this parameter might be a suitable means of predicting TG-induced toxicity in vivo. We propose that the low background expected for this kind of measurement would make it particularly appropriate for the analysis of clinical specimens (e.g., mononuclear bone marrow cells) from leukemic patients receiving thiopurines, to monitor (and, hopefully, predict) their response to treatment.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46920/1/280_2004_Article_BF00304760.pd

Penetration of topical diclofenac into synovial tissue and fluid of osteoarthritic knees: a multicenter, randomized, placebo-controlled, pharmacokinetic study

Funder: GSK Consumer Healthcare S.A., Nyon, SwitzerlandBackground:: Topical diclofenac, a nonsteroidal anti-inflammatory drug, has proven efficacy and safety in the management of osteoarthritis pain. We investigated penetration of topical diclofenac into knee synovial tissue and fluid (primary objective) and evaluated relative exposure in the knee versus plasma (secondary objective). Methods:: In this phase I, double-blind, multicenter study, patients scheduled for arthroplasty for end-stage knee osteoarthritis were randomly assigned 2:1 to 4 g diclofenac diethylamine 2.32% w/w gel (92.8 mg diclofenac diethylamine, equivalent to 74.4 mg diclofenac, per application) or placebo gel, applied to the affected knee by a trained nurse/designee every 12 h for 7 days before surgery. Diclofenac concentrations were measured in synovial tissue, synovial fluid and plasma from samples obtained during surgery ⩾12 h after last application. Treatment-emergent adverse events (TEAEs) were evaluated. Results:: Evaluable synovial tissue or fluid samples were obtained from 45 (diclofenac n = 29; placebo n = 16) of 47 patients. All diclofenac-treated participants had measurable diclofenac concentrations in synovial tissue [geometric mean 1.57 (95% confidence interval (CI) 1.12, 2.20) ng/g] and fluid [geometric mean 2.27 (95% CI 1.87, 2.76) ng/ml] ⩾12 h after the last dose. Geometric mean (95% CI) ratio of diclofenac in synovial tissue:plasma was 0.32 (0.23, 0.45) and in synovial fluid:plasma was 0.46 (0.40, 0.54). TEAE rates were similar for diclofenac (55.2%) and placebo (58.8%); none were treatment related. Conclusions:: Topical diclofenac diethylamine 2.32% w/w gel penetrated into the osteoarthritic knee after repeated application and remained detectable in synovial tissue and fluid at the end of the final 12 h dosing cycle

Simulation of dilated heart failure with continuous flow circulatory support

Lumped parameter models have been employed for decades to simulate important hemodynamic couplings between a left ventricular assist device (LVAD) and the native circulation. However, these studies seldom consider the pathological descending limb of the Frank-Starling response of the overloaded ventricle. This study introduces a dilated heart failure model featuring a unimodal end systolic pressure-volume relationship (ESPVR) to address this critical shortcoming. The resulting hemodynamic response to mechanical circulatory support are illustrated through numerical simulations of a rotodynamic, continuous flow ventricular assist device (cfVAD) coupled to systemic and pulmonary circulations with baroreflex control. The model further incorporated septal interaction to capture the influence of left ventricular (LV) unloading on right ventricular function. Four heart failure conditions were simulated (LV and bi-ventricular failure with/ without pulmonary hypertension) in addition to normal baseline. Several metrics of LV function, including cardiac output and stroke work, exhibited a unimodal response whereby initial unloading improved function, and further unloading depleted preload reserve thereby reducing ventricular output. The concept of extremal loading was introduced to reflect the loading condition in which the intrinsic LV stroke work is maximized. Simulation of bi-ventricular failure with pulmonary hypertension revealed inadequacy of LV support alone. These simulations motivate the implementation of an extremum tracking feedback controller to potentially optimize ventricular recovery. © 2014 Wang et al

Pemetrexed pharmacokinetics and pharmacodynamics in a phase I/II study of doublet chemotherapy with vinorelbine: implications for further optimisation of pemetrexed schedules

The purpose of this study was to investigate the utility of plasma pharmacokinetic and pharmacodynamic measures including plasma deoxynucleosides, homocysteine and methylmalonic acid concentrations in understanding the time course and extent of the inhibition of thymidylate synthase (TS) by pemetrexed in the context of a phase I/II combination study with vinorelbine. Eighteen patients received supplementation with folic acid and Vitamin B12 1 week before beginning treatment with pemetrexed and vinorelbine administered in a dose-escalating manner on a 21-day cycle. Heparinised blood samples were collected from consenting patients in the first cycle for pharmacokinetic analyses and in the first two cycles for determination of plasma thymidine, deoxyuridine, homocysteine and methylmalonic acid concentrations. These values were correlated with response and toxicity. Plasma deoxyuridine was used as a measure of TS inhibition, and concentrations of deoxyuridine were significantly elevated relative to baseline on days 1 (P<0.01), 2 (P<0.001) and 3 (P<0.05) after treatment at all pemetrexed dose levels (400–700 mg m−2). The magnitude of deoxyuridine elevation correlated with pemetrexed area under the plasma concentration–time curve (AUC) (r2=0.23, P<0.05). However, deoxyuridine concentrations returned to baseline between 8 and 15 days after treatment with pemetrexed, suggesting that inhibition of TS was not durable. Pemetrexed AUC correlated with the percentage decline (relative to baseline) in both platelets (r2=0.58, P<0.001) and leucocytes (r2=0.26, P<0.05) at day 8. Baseline homocysteine was also significantly correlated with these measures of haematological toxicity (r2=0.37, P<0.01 and r2=0.39, P<0.01, respectively). In addition, there was a significant reduction of plasma homocysteine on days 8 (P<0.005) and 15 (P<0.05) in cycle 1 compared to baseline values. The results suggest that the TS inhibitory effects of pemetrexed are short-lived and make the case for a more frequent schedule of administration such as every 2 weeks. The lack of protracted TS inhibition may be due to concomitant vitamin administration, and this may be the mechanism by which vitamins prevent life-threatening toxicity from pemetrexed. Baseline homocysteine concentration remains a predictive marker for haematological toxicity even following folate supplementation

Response of human HT-29 colorectal tumor cells to extended exposure to bromodeoxyuridine

Effects of the extended exposure of a human colorectal tumor-cell line (HT-29) to bromodeoxyuridine (BrdUrd) were studied in anticipation of the clinical use of that agent to treat colorectal cancer, particularly as a regionally delivered radiosensitizer. We found that 72-h exposure to a concentration of BrdUrd that is estimated to be locally maintained in the liver (100 μ M ) was significantly cytotoxic with a 3-log reduction in survival. As measured by GC/MS-SIM method, incorporation of BrdUrd into DNA followed an unexpected time course in that continuous exposure to 10 μ M BrdUrd resulted in maximal incorporation at 3 days, after which the extent of incorporated analog fell significantly (despite daily changes of the medium). This finding was apparently due to a greater rate of loss of BrdUrd from the medium at later time points. Flow cytometric analysis using an anti-BrdUrd antibody (IU-4) revealed that antibody binding also peaked and fell off with time. However, at exposure times of >24 h, the timing and extent of this decline were significantly different than had been indicated by the GC/MS method. These results indicate that the quantitative relationship between antibody staining and BrdUrd incorporation changes as drug-exposure time increases and that quantitative studies of anti-BrdUrd antibody binding must be interpreted with caution, especially when extended drug-treatment protocols have been used.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46921/1/280_2004_Article_BF00694337.pd

Die Entwickelung des altisraelitischen Priesterthums : ein Beitrag zur Kritik der mittleren Bücher des Pentateuchs

von S. Maybau