Biomedical subjects

Robert A Dean

Publications and source records attributed to Robert A Dean.

4 recordsLinked to original sources

PubMed2006

Quantitative analysis of amyloid beta peptides in cerebrospinal fluid of Alzheimer's disease patients by immunoaffinity purification and stable isotope dilution liquid chromatography/negative electrospray ionization tandem mass spectrometry.

The 40 and 42 amino-acid residue forms of amyloid beta (Abeta(1-40) and Abeta(1-42)) in cerebrospinal fluid (CSF) have been proposed as potential biomarkers of Alzheimer's disease (AD). Quantitative analyses of Abeta peptides in CSF have relied almost exclusively on the use of immunoassay-based assays such as the enzyme-linked immunosorbent assay (ELISA) procedure. However, due to the ability of the Abeta peptides to readily self-aggregate or bind to other proteins and glassware, such analyses are extremely challenging. Analyses are further complicated by the potential of the peptides to undergo post-translational modifications and the possibilities for cross-reaction in the ELISA assays with endogenous components of the CSF. An approach based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) has now been developed which overcomes these methodological issues. The key steps in implementing this new approach involved immunoaffinity purification coupled with the use of [15N]-labeled Abeta peptides as internal standards, a basic LC mobile phase, negative ion electrospray ionization, and a basic solvent for dissolving the peptides and washing the injection needle to prevent carryover of analytes during multiple injections on the LC/MS system. The validated method had limits of quantitation of 44 fmol/mL (200 pg/mL) for Abeta(1-42) and 92 fmol/mL (400 pg/mL) for Abeta(1-40). An excellent correlation was found between the LC/MS/MS assay and an ELISA assay for Abeta(1-42) in human CSF (r2 = 0.915), although less correlation was observed for Abeta(1-40) (r2 = 0.644). Mean CSF Abeta(1-42) concentrations for samples collected 2 weeks apart from a limited number of AD patients provided additional confidence in the reproducibility of the LC/MS/MS assay. Concentrations for duplicate samples from AD patients were slightly higher than most previously reported values (mean 1.06 +/- 0.25 ng/mL; n = 7). Abeta(1-40) concentrations in duplicate samples obtained from AD patients were also reproducible but were found to be slightly lower than most previously reported values (mean 6.36 +/- 3.07 ng/mL; n = 7). Consistent with literature reports, mean Abeta(1-42) concentrations were found to be lower in AD patients compared with the normal subjects (mean 1.49 +/- 0.59 ng/mL; n = 7), whereas there was no difference in Abeta(1-40) concentrations between AD patients and normal subjects (mean 5.88 +/- 3.03 ng/mL; n = 7). The accuracy and precision of the LC/MS assay mean that it will be a useful complement to existing ELISA assays for monitoring therapeutic interventions designed to modulate CSF Abeta(1-42) concentrations in individual AD patients. Moreover, the introduction of stable isotope labeled internal standards offers the potential to achieve a more rigorous account of the influence of methodological effects related to sample collection and processing.

Alzheimer Disease↗

PubMed2006

New pathways in drug discovery for Alzheimer's disease.

Specific treatments for Alzheimer's disease (AD) were first introduced in the 1990s using the acetyl-cholinesterase inhibitors. More recently, the N-methyl-D-aspartate (NMDA) antagonist memantine has become available. Although these treatments do provide a modest improvement in the cognitive abnormalities present in AD, their pharmacology is based on manipulation of neurotransmitter systems, and there is no compelling evidence that they interfere with the underlying pathogenic process. Pathologic and genetic data have led to the hypothesis that a peptide called amyloid ss(Abeta) plays a primary role in the pathophysiology of AD. Several investigational therapies targeting Abeta are now undergoing clinical trials. This paper reviews the available data regarding Abeta-directed therapies that are in the clinic and summarizes the approach to biomarkers and clinical trial designs that can provide evidence of modification of the underlying disease process.

Alzheimer Disease↗

PubMed2005

Development, validation, and implementation of a multiplex immunoassay for the simultaneous determination of five cytokines in human serum.

Quantification of biomarkers can provide important information about the safety and efficacy of candidate drugs. Unfortunately, limited sample volume and excess costs often limit analysis of multiple biomarkers. We developed, optimized, validated, and implemented a multiplex immunoassay for simultaneous measurement of multiple circulating cytokines: IL-1beta, TNFalpha, IL-6, IL-8, and IL-10. Multiplex immuoassays were performed using the Luminex LabMAP instrument. Capture antibodies for each cytokine were covalently bound to distinct microsphere subsets distinguished by differing dye ratios. The concentration of each individual cytokine determined by measuring orange fluorescence produced by a complex of a biotinylated cytokine-specific antibody and streptavidin-phycoerythrin. The lower limit of quantification for all assays was 20 pg/mL with the exception of IL-8 which was 100 pg/mL. The inter-assay precision was less than 25%CV for all analytes at all control levels both pre-study and in-study. The percent recovery ranged from 83 to 108% pre-study and 90 to 125% in-study. In a linearity assessment, a 15,000 pg/mL multi-analyte control could be diluted 1:50 and maintain expected accuracy. We measured the cytokine concentrations in more than 2000 serum samples from patients with sepsis. Multiplex results for IL-6 were compared to a conventional commercially available ELISA kit. The degree of agreement between the two methods as measured by the concordance correlation coefficient was 84.5%. Multiplex results were 2.36-fold higher than ELISA values on the average. After adjusting for this mean difference, the 95% empirical limits of agreement for the ratio of individual sample values were 0.33, 2.65. This multiplex immunoassay provided simultaneous measurement of circulating cytokines using 80% less patient specimen compared to traditional approaches and at a significantly decreased cost. Efficient use of this platform requires process improvements to fully maximize the positive impact of multiplex assays in clinical drug development.

Cytokines↗

PubMedDate not supplied

Safety, tolerability, and changes in amyloid beta concentrations after administration of a gamma-secretase inhibitor in volunteers.

Amyloid beta (Abeta) may play a central role in the pathogenesis of Alzheimer disease. A functional gamma-secretase inhibitor, LY450139, was developed that inhibits Abeta formation in whole cell assays, transgenic mice, and beagle dogs. The authors wished to determine the safety and tolerability of this drug, and the reduction of Abeta in plasma and cerebrospinal fluid (CSF) after multiple doses. Volunteer subjects (N = 37) were studied using doses from 5 to 50 mg/day given for 14 days. Plasma and CSF concentrations of LY450139, Abeta(1-40) and Abeta(1-X) ("Abeta(total)") were determined, and safety and tolerability were assessed. The plasma half-life of LY450139 was approximately 2.5 hours. Pharmacokinetic analyses showed a linear relationship between dose and plasma concentrations, with a Cmax of 828 +/- 19.2 ng/mL after a 50-mg dose. Plasma Abeta concentrations decreased in a dose-dependent manner over a 6-hour interval following drug administration, with a maximum decrease of approximately 40% relative to baseline. After returning to baseline, Abeta concentrations were transiently increased. CSF Abeta concentrations were unchanged. Adverse events reported by subjects taking 5-mg, 20-mg, or 40-mg doses were similar to those reported by subjects taking placebo. Two of 7 subjects taking 50 mg/day experienced adverse events that may have been drug related. In this phase 1 volunteer study, reported adverse events after taking LY450139 were manageable. A dose-dependent reduction in plasma Abeta was demonstrated, and changes in plasma Abeta concentrations were temporally related to the pharmacokinetic characteristics of LY450139.

Aged↗