Leona Fields

Immunosuppressive tor kinase inhibitor everolimus (RAD) suppresses growth of cells derived from posttransplant lymphoproliferative disorder at allograft-protecting doses

Background. Posttransplant lymphoproliferative disorders (PTLDs) represent a life-threatening complication of standard immunosuppressive therapy. The impact of novel, rapamycin-related immunosuppressive drugs on the pathogenesis of PTLDs remains undefined. Methods. We tested the effect of everolimus (RAD, Novartis Pharma AG, Basel, Switzerland) on human PTLD-derived cells using in vitro assays and an in vivo severe combined immunodeficiency disease mouse xenotransplant model. Results. Everolimus profoundly inhibited the proliferation, cell-cycle progression, and survival of the PTLD-1 cell line established from a pulmonary PTLD. Equally profound inhibition of PTLD-1 growth was achieved in vivo at well-tolerated everolimus doses of 0.5 to 5 mg/kg per day. Five mg/kg per day of everolimus, given once per day, inhibited PTLD-1 tumor volume gain by more than 10-fold in treated mice compared with untreated mice. Because the subsequent pharmacokinetic analysis indicated rapid everolimus absorption, distribution, and clearance in mice (with a half-life of 3 to 6 hr and maximum drug blood concentration reached after 0.5 to 1 hr), treatment was changed to a twice-daily regimen. Everolimus given twice daily at 0.5 mg/kg per dose inhibited tumor-volume gain by more than 60-fold and at 0.25 mg/kg per dose by more than 10-fold. Similar everolimus doses were required to prevent graft rejection in a mouse heart allotransplantation model; the highest dose tested (1.5 mg/kg twice daily) resulted in long-term graft survival in all mice that underwent transplantation. Conclusions. Everolimus displays a potent inhibitory effect on PTLD-derived cells in vitro and in vivo in a dose range leading to prevention of allograft rejection and may prove effective in both the prevention and treatment of PTLDs in transplant patients.

Factors influencing theophylline serum protein binding.

We studied a number of influences on theophylline binding to serum proteins using equilibrium dialysis (37°), a modified Krebs‐Ringer bicarbonate buffer (pH 7.4), and 8‐14C‐theophylline with unlabeled theophylline (30 μg/ml) added to sera from healthy subjects. Theophylline protein binding rose by 18.6% as pH rose from 7.0 to 7.8 (percent theophylline bound = 28.2 ±4.3 at pH 7.0 and 46.8 ± 4.9 at pH 7.8, n = 5). Average theophylline binding to the proteins at 37° in serum samples from 10 normal adults was 39.3 ±3.44%, which is 8.9% lower than the average of 48.2 ± 3.74% for the same samples at 26°. Theophylline binding was 6.1% higher with 0.1 mole/l phosphate buffer, pH 7.4, than with a modified Krebs‐Ringer bicarbonate buffer, pH 7.4. Of the 19 drugs and metabolites tested for competition with theophylline for binding sites on serum proteins, 10 induced decreases in binding ranging from 6.8% in the case of furosemide to 18.3% for sodium salicylate. The latter was the only drug that induced a decrease in theophylline binding at concentrations that would be achieved in the therapy of some patients (i.e., patients on long‐term salicylate therapy). All the other drugs that decreased theophylline binding did so at much greater concentrations than their usual therapeutic levels. The mean ± SD of theophylline bound in 51 fresh serum samples from healthy adults was 48.6 ± 10.2%; the pH of these specimens varied from 7.6 to 8.7. After adjusting pH to 7.4, theophylline binding was lowered to 37.6 ± 4.5% and intersubject variability decreased. We recommend that the pH of serum specimens be adjusted to 7.4, or to the original pH of the blood specimen if it differs significantly from 7.4 (i.e., in acidotic or alkalotic patients). The wide range of reported values for theophylline binding to serum proteins in normal and asthmatic adults at least partly results from differences in the conditions used for the separation of free from bound drug.

Cocaethylene Levels in Patients Who Test Positive for Cocaine

STUDY OBJECTIVE To determine the presence of cocaethylene, an active metabolite of the combination of cocaine and ethanol, among trauma patients who test positive for cocaine. METHODS We assembled a case series of 416 consecutive urban trauma center patients with major trauma. Urine was tested for the presence of the cocaine metabolite benzoylecgonine. Plasma was quantitatively assayed for cocaine, ethanol, and cocaethylene. RESULTS Of the study subjects, 158 (38%) were positive for benzoylecgonine. Of the 114 of these subjects who had adequate plasma specimens, 68 (60%) tested positive for cocaethylene (mean, 41 +/- 27 ng/mL; range, 3 to 213 ng/mL), all tested positive for cocaine (mean, 92.9 +/- 52 ng/mL), and 56% were positive for ethanol (mean, 175 +/- 85 mg/mL). We found poor correlation between admission levels of cocaethylene and cocaine (R=.02), even when subjects were stratified by ethanol level. The correlation between cocaethylene and ethanol levels was weak (R=.24). Of the 68 patients who tested positive for cocaethylene, 29% tested negative for ethanol. Plasma was also assayed from 94 subjects who tested negative for benzoylecgonine; 9% had detectable levels of cocaine, and 2% had detectable levels of cocaethylene. CONCLUSION Cocaethylene was present in more than half of the subjects who tested positive for cocaine.

The Alzheimer’s Disease Neuroimaging Initiative 2 Biomarker Core: A review of progress and plans

We describe Alzheimers Disease Neuroimaging Initiative (ADNI) Biomarker Core progress including: the Biobank; cerebrospinal fluid (CSF) amyloid beta (Aβ1–42), t‐tau, and p‐tau181 analytical performance, definition of Alzheimers disease (AD) profile for plaque, and tangle burden detection and increased risk for progression to AD; AD disease heterogeneity; progress in standardization; and new studies using ADNI biofluids.

High-performance liquid chromatography-mass spectroscopy/mass spectroscopy method for simultaneous quantification of total or free fraction of mycophenolic acid and its glucuronide metabolites.

Measurement of unbound fractions of mycophenolic acid and its metabolites may prove useful in explaining the complicated pharmacokinetic and pharmacodynamic behavior of this drug as well as in therapeutic drug monitoring. We developed a reliable, accurate, and sensitive liquid chromatography-tandem mass spectrometric method for the simultaneous quantification of mycophenolic acid (MPA), MPA glucuronide (MPAG), and MPA acyl-glucuronide (AcMPAG), total or unbound, in plasma, urine, and tissue extract. This method uses a single internal standard, carboxy-butoxy ether of mycophenolic acid (MPAC), and involves a simple sample preparation step. Aliquots of plasma, urine, or dissolved tissue extract (100 μL) or plasma ultrafiltrate for free analytes (50 μL) are treated with acetonitrile/formic acid mixture (99.5/0.5 v/v) followed by centrifugation and dilution with water. The prepared samples are then injected onto an extraction column (Eclipse XDB-C18 12.5 × 4.1 mm; Agilent Technologies, Palo Alto, CA) and washed with mobile phase composed of acetonitrile/water/formic acid (10/89.5/0.5 v/v/v) at a flow rate of 2.8 mL/min. A switching valve is activated 1 minute after sample injection. The analytes are eluted onto the analytical column (Eclipse XDB-C18 150 × 4.1 mm; Agilent Technologies) with a gradient of 0.5% aqueous formic acid, methanol, acetonitrile, and water. We used a tandem mass spectrometer with electrospray ion source, in which the tandem mass spectroscopy transitions were (m/z): 338→207 for MPA, 438→303 for MPAC, and 514→303 for MPAG and AcMPAG. The dynamic ranges (lower limit of quantitation and upper limit of quantitation) were as follows: 0.05 to 30 mg/L for total MPA and 1 to 300 μg/L for free MPA; 0.5 to 300 mg/L of total MPAG and 0.2 to 60 mg/L for free MPAG; and 0.025 to 15 mg/L of total AcMPAG and 1 to 60 μg/L for free AcMPAG. The precision at lower limit of quantitation was in the range of 8.0% to 11.9% for all three total analytes and 13.8 to 18.7% for the free analytes. Accuracy at lower limit of quantitation was in the range of 100% to 105% for total and 97% to 99% for free analytes. Between-day precision of quality control samples was 4.0% to 6.3% for human plasma spiked with total analytes and 4.5% to 14.4% for spiked plasma ultrafiltrate for free analytes. Mean absolute recovery ranged from 98.5% to 101.7% for MPA (both total and free), from 78.1% to 103.4% for MPAG and from 91.5% to 110.4% for AcMPAG. No significant ion suppression was found under these conditions for any of the analytes. Carryover effect was found to be at a maximum level of 0.02%. This method was successfully applied to analyze over 11,000 samples for total analytes, and over 8000 samples for free analytes in plasma, and has been in operation for nearly 3 years without loss of performance.

METHOD COMPARISON OF AB(1-42) MEASURED IN HUMAN CEREBROSPINAL FLUID SAMPLES BY LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY, THE INNO-BIA ALZBIO3 ASSAY, AND THE ELECSYS® B-AMYLOID(1-42) ASSAY

INHUMANCEREBROSPINALFLUID SAMPLESBY LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY, THE INNO-BIA ALZBIO3 ASSAY, AND THE ELECSYS B-AMYLOID(1-42) ASSAY LeslieM. Shaw, Leona Fields, Magdalena Korecka, TeresaWalig orska, John Q. Trojanowski, Deirdre Allegranza, Tobias Bittner, Ying He, Kelly Morgan, Christina Rabe, University of Pennsylvania Hospital, Philadelphia, PA, USA; 2 Roche Diagnostics International AG, Rotkreuz, Switzerland; 3 Roche Diagnostics GmbH, Penzberg, Germany; 4 Roche Diagnostics Corporation, Indianapolis, IN, USA. Contact e-mail: Les. Shaw@uphs.upenn.edu

The use of concentration measurements of parent drug and metabolites during clinical trials.

The need for well-designed pharmacokinetic (PK) and pharmaco-dynamic (PD) studies early in the development of new drugs is described. In this review we illustrate the application and cost-effectiveness of optimal sampling theory in PK study design for ongoing clinical trial studies of ethyol, a chemoprotector drug. The importance of careful selection of the appropriate biological fluid in which to measure drug concentration at the earliest possible stage of new drug development is described in the context of the development of new immunosuppressive drugs. We focus on the requirement for well-validated analytical methodology in PK-PD studies, described in a discussion of the analytical methodology in use in clinical trials of two immunosuppressive agents, cyclosporin G and RS-61443 (mycophenolate mofetil).

Prospective quality-control monitoring in the context of a clinical trial

enforce boundary smoothness. The technique’s validity was tested by comparing our results to the manual HarP reference segmentations on a sample of 100 subjects from the ADNeuroimaging Initiative (ADNI) database. We used a leave-one-out strategy to train and then test the segmentations. We used Dice similarity index for objects and correlation coefficients for volumes to verify compliance of automated to manual segmentations. Results: The results show that our method is bias-free with an average Dice similarity coefficient value of 0.803 and overall correlation coefficient of r1⁄40.95. Figure 1 compares the HC volumes computed from the segmentations made by anatomists (reference volumes) with the corresponding volumes computed from the automated segmentations (estimated segmentations). Two fits are shown, with and without intercept (fit parameters listed in Table 1). Both fits are virtually indistinguishable. Overlaid to a MRI image, the new contours often outperform those made by the anatomists in delineating the HC tissues in all three planes, due to the increased smoothness. Conclusions:Our automated segmentation algorithm was able to generate accurate HC volume measurements on a sample of the ADNI cohort that is heterogeneous in terms of ages, cognitive status, manufacturers, and atrophy levels. To our knowledge, this is the first study to assess the accuracy of an automated algorithm with the official release of HarP segmentation labels. Our results hold promise for the utilisation of automated segmentation in large clinical trials and in clinical practice where there is a growing need for biomarkers to support diagnosis and monitor progression of AD.

NOVEL CSF BIOMARKERS OF NEURONAL INJURY, SYNAPTIC DYSFUNCTION AND NEUROINFLAMMATION IN AUTOSOMAL DOMINANT ALZHEIMER DISEASE: VILIP-1, NEUROGRANIN, SNAP-25 AND YKL-40 IN THE DOMINANTLY INHERITED ALZHEIMER NETWORK (DIAN)

O3-14-01 NOVEL CSF BIOMARKERS OF NEURONAL INJURY, SYNAPTIC DYSFUNCTION AND NEUROINFLAMMATION IN AUTOSOMAL DOMINANTALZHEIMER DISEASE: VILIP-1, NEUROGRANIN, SNAP-25 AND YKL-40 IN THE DOMINANTLY INHERITED ALZHEIMER NETWORK (DIAN) Anne M. Fagan, Yan Li, Kaitlin Todd, Elizabeth M. Herries, Rachel L. Henson, Suzanne E. Schindler, Julia D. Gray, GuoqiaoWang, Danielle Graham, Leona Fields, Leslie M. Shaw, Jack H. Ladenson, Jason Hassenstab, Tammie L. S. Benzinger, John C. Morris, Randall J. Bateman, Chengjie Xiong, Washington University School of Medicine, Saint Louis, MO, USA; Washington University School of Medicine, St. Louis, MO, USA; Biogen, Cambridge, MA, USA; University of Pennsylvania, Philadelphia, PA, USA; Washington University in St. Louis School of Medicine, St. Louis, MO, USA. Contact e-mail: fagana@ wustl.edu

Derivation of cutoffs for the Elecsys® amyloid β (1–42) assay in Alzheimer's disease

An Elecsys® Amyloid β (Aβ [1–42]) immunoassay cutoff for classification of patients with Alzheimers disease was investigated.