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

Analysis of bromodeoxyuridine incorporation into DNA: Comparison of gas chromatographic/mass spectrometric, CsCl gradient sedimentation, and specific radioactivity methods

A sensitive new method for the quantitation of 5-bromodeoxyuridine (BrdUrd) incorporated into DNA by GC/MS analysis of enzymatically released Thy and bromouracil (BrUra) is presented. The hydrolysis procedure was characterized and found to give uniform results when sample size was 1-10 [mu]g DNA and incubation time for DNA digestion was between 40 min and 16 h. Samples of DNA containing 3H-labeled BrdUrd were analyzed in parallel by the GC/MS technique and by specific radioactivity and buoyant density measurements, in order to compare the three methods. The GC/MS procedure gave values for percentage replacement of Thy by BrUra which were higher than those obtained by specific activity and lower than those obtained by buoyant density. This GC/MS method can detect 1% replacement in a 1-[mu]g DNA sample, equivalent to approximately 105 cells or 0.1 mg tissue, and will permit sensitive and quantitative analysis of the presence of this chemotherapeutic agent/radiosensitizer in cellular DNA from biopsy samples of normal or tumor tissue.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26801/1/0000357.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

Mechanistic mathematical modelling of mercaptopurine effects on cell cycle of human acute lymphoblastic leukaemia cells

The antimetabolite mercaptopurine (MP) is widely used to treat childhood acute lymphoblastic leukaemia (ALL). To study the dynamics of MP on the cell cycle, we incubated human T-cell leukaemia cell lines (Molt-4 sensitive and resistant subline and P12 resistant) with 10 μM MP and measured total cell count, cell cycle distribution, percent viable, percent apoptotic, and percent dead cells serially over 72 h. We developed a mathematical model of the cell cycle dynamics after treatment with MP and used it to show that the Molt-4 sensitive controls had a significantly higher rate of cells entering apoptosis (2.7-fold, P<0.00001) relative to the resistant cell lines. Additionally, when treated with MP, the sensitive cell line showed a significant increase in the rate at which cells enter apoptosis compared to its controls (2.4-fold, P<0.00001). Of note, the resistant cell lines had a higher rate of antimetabolite incorporation into the DNA of viable cells (>1.4-fold, P<0.01). Lastly, in contrast to the other cell lines, the Molt-4 resistant subline continued to cycle, though at a rate slower relative to its control, rather than proceed to apoptosis. This led to a larger S-phase block in the Molt-4 resistant cell line, but not a higher rate of cell death. Gene expression of apoptosis, cell cycle, and repair genes were consistent with mechanistic dynamics described by the model. In summary, the mathematical model provides a quantitative assessment to compare the cell cycle effects of MP in cells with varying degrees of MP resistance

Myostatin Is Elevated in Congenital Heart Disease and After Mechanical Unloading

Myostatin is a negative regulator of skeletal muscle mass whose activity is upregulated in adult heart failure (HF); however, its role in congenital heart disease (CHD) is unknown.We studied myostatin and IGF-1 expression via Western blot in cardiac tissue at varying degrees of myocardial dysfunction and after biventricular support in CHD by collecting myocardial biopsies from four patient cohorts: A) adult subjects with no known cardiopulmonary disease (left ventricle, LV), (Adult Normal), (n = 5); B) pediatric subjects undergoing congenital cardiac surgery with normal RV size and function (right ventricular outflow tract, RVOT), (n = 3); C) pediatric subjects with worsening but hemodynamically stable LV failure [LV and right ventricle (LV, RV,)] with biopsy collected at the time of orthotopic heart transplant (OHT), (n = 7); and D) pediatric subjects with decompensated bi-ventricular failure on BiVAD support with biopsy collected at OHT (LV, RV, BiVAD), (n = 3).The duration of HF was longest in OHT patients compared to BIVAD. The duration of BiVAD support was 4.3±1.9 days. Myostatin expression was significantly increased in LV-OHT compared to RV-OHT and RVOT, and was increased more than double in decompensated biventricular HF (BiVAD) compared to both OHT and RVOT. An increased myostatin/IGF-1 ratio was associated with ventricular dysfunction.Myostatin expression in increased in CHD, and the myostatin/IGF-1 ratio increases as ventricular function deteriorates. Future investigation is necessary to determine if restoration of the physiologic myostatin/IGF-1 ratio has therapeutic potential in HF

Defining levels of care in cardiogenic shock

BackgroundExpert opinion and professional society statements have called for multi-tier care systems for the management of cardiogenic shock (CS). However, little is known about how to pragmatically define centers with different levels of care (LOC) for CS.MethodsEleven of 23 hospitals within our healthcare system sharing a common electronic health record were classified as different LOC according to their highest mechanical circulatory support (MCS) capabilities: Level 1 (L-1)—durable left ventricular assist device, Level 1A (L-1A)—extracorporeal membrane oxygenation, Level 2 (L-2)—intra-aortic balloon pump and percutaneous ventricular assist device; and Level 3 (L-3)—no MCS. All adult patients treated for CS (International Classification of Diseases, ICD-10 code R57.0) between 2016 and 2022 were included. Etiologies of CS were identified using associated diagnostic codes. Management strategies and outcomes across LOC were compared.ResultsHigher LOC centers had higher volumes: L-1 (n = 1): 2,831 patients, L-1A (n = 4): 3,452, L-2 (n = 1): 340, and L-3 (n = 5): 780. Emergency room admissions were more common in lower LOC (96% at L-3 vs. 46% L-1; p &lt; 0.001), while hospital transfers were predominant at higher LOC (40% at L-1 vs. 2.7% at L-3; p &lt; 0.001). Men comprised 61% of the cohort. Patients were younger in the higher LOC [69 (60–78) years at L-1 vs. 77 (67–85) years at L-3; p &lt; 0.001]. Patients with acute myocardial infarction (AMI)-CS and acute heart failure (AHF)-CS were concentrated in higher LOC centers while other etiologies of CS were more common in L-2 and L-3 (p &lt; 0.001). Cardiac arrest on admission was more prevalent in lower LOC centers (L-1: 2.8% vs. L-3: 12.1%; p &lt; 0.001). Patients with AMI-CS received more percutaneous coronary intervention in lower LOC (51% L-2 vs. 29% L-1; p &lt; 0.01) but more coronary arterial bypass graft surgery at higher LOC (L-1: 42% vs. L-1A: 23%; p &lt; 0.001). MCS use was consistent across levels for AMI-CS but was more frequent in higher LOC for AHF-CS patients (L-1: 28% vs. L-2: 10%; p &lt; 0.001). Despite increasing in-hospital mortality with decreasing LOC, no significant difference was seen after multivariable adjustment.ConclusionThis is the first report describing a pragmatic classification of LOC for CS which, based on MCS capabilities, can discriminate between centers with distinct demographics, practice patterns, and outcomes. This classification may serve as the basis for future research and the creation of CS systems of care

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

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

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