Flora Berisha

Cerebrospinal fluid neurofilament concentration reflects disease severity in frontotemporal degeneration

Cerebrospinal fluid (CSF) neurofilament light chain (NfL) concentration is elevated in neurological disorders, including frontotemporal degeneration (FTD). We investigated the clinical correlates of elevated CSF NfL levels in FTD.

Characterization of Novel CSF Tau and ptau Biomarkers for Alzheimer’s Disease

Cerebral spinal fluid (CSF) Aβ42, tau and p181tau are widely accepted biomarkers of Alzheimer’s disease (AD). Numerous studies show that CSF tau and p181tau levels are elevated in mild-to-moderate AD compared to age-matched controls. In addition, these increases might predict preclinical AD in cognitively normal elderly. Despite their importance as biomarkers, the molecular nature of CSF tau and ptau is not known. In the current study, reverse-phase high performance liquid chromatography was used to enrich and concentrate tau prior to western-blot analysis. Multiple N-terminal and mid-domain fragments of tau were detected in pooled CSF with apparent sizes ranging from <20 kDa to ~40 kDa. The pattern of tau fragments in AD and control samples were similar. In contrast, full-length tau and C-terminal-containing fragments were not detected. To quantify levels, five tau ELISAs and three ptau ELISAs were developed to detect different overlapping regions of the protein. The discriminatory potential of each assay was determined using 20 AD and 20 age-matched control CSF samples. Of the tau ELISAs, the two assays specific for tau containing N-terminal sequences, amino acids 9-198 (numbering based on tau 441) and 9-163, exhibited the most significant differences between AD and control samples. In contrast, CSF tau was not detected with an ELISA specific for a more C-terminal region (amino acids 159-335). Significant discrimination was also observed with ptau assays measuring amino acids 159-p181 and 159-p231. Interestingly, the discriminatory potential of p181 was reduced when measured in the context of tau species containing amino acids 9-p181. Taken together, these results demonstrate that tau in CSF occurs as a series of fragments and that discrimination of AD from control is dependent on the subset of tau species measured. These assays provide novel tools to investigate CSF tau and ptau as biomarkers for other neurodegenerative diseases.

Recommendations for Use and Fit-for-Purpose Validation of Biomarker Multiplex Ligand Binding Assays in Drug Development

Multiplex ligand binding assays (LBAs) are increasingly being used to support many stages of drug development. The complexity of multiplex assays creates many unique challenges in comparison to single-plexed assays leading to various adjustments for validation and potentially during sample analysis to accommodate all of the analytes being measured. This often requires a compromise in decision making with respect to choosing final assay conditions and acceptance criteria of some key assay parameters, depending on the intended use of the assay. The critical parameters that are impacted due to the added challenges associated with multiplexing include the minimum required dilution (MRD), quality control samples that span the range of all analytes being measured, quantitative ranges which can be compromised for certain targets, achieving parallelism for all analytes of interest, cross-talk across assays, freeze-thaw stability across analytes, among many others. Thus, these challenges also increase the complexity of validating the performance of the assay for its intended use. This paper describes the challenges encountered with multiplex LBAs, discusses the underlying causes, and provides solutions to help overcome these challenges. Finally, we provide recommendations on how to perform a fit-for-purpose-based validation, emphasizing issues that are unique to multiplex kit assays.

The γ-Secretase Modulator, BMS-932481, Modulates Aβ Peptides in the Plasma and Cerebrospinal Fluid of Healthy Volunteers.

The pharmacokinetics, pharmacodynamics, safety, and tolerability of BMS-932481, a γ-secretase modulator (GSM), were tested in healthy young and elderly volunteers after single and multiple doses. BMS-932481 was orally absorbed, showed dose proportionality after a single dose administration, and had approximately 3-fold accumulation after multiple dosing. High-fat/caloric meals doubled the Cmax and area under the curve and prolonged Tmax by 1.5 hours. Consistent with the preclinical pharmacology of GSMs, BMS-932481 decreased cerebrospinal fluid (CSF) Aβ39, Aβ40, and Aβ42 while increasing Aβ37 and Aβ38, thereby providing evidence of γ-secretase enzyme modulation rather than inhibition. In plasma, reductions in Aβ40 and Aβ42 were observed with no change in total Aβ; in CSF, modest decreases in total Aβ were observed at higher dose levels. Increases in liver enzymes were observed at exposures associated with greater than 70% CSF Aβ42 lowering after multiple dosing. Although further development was halted due to an insufficient safety margin to test the hypothesis for efficacy of Aβ lowering in Alzheimer’s disease, this study demonstrates that γ-secretase modulation is achievable in healthy human volunteers and supports further efforts to discover well tolerated GSMs for testing in Alzheimer’s disease and other indications.

Robust Translation of γ-Secretase Modulator Pharmacology across Preclinical Species and Human Subjects

The amyloid-β peptide (Aβ)—in particular, the 42–amino acid form, Aβ1-42—is thought to play a key role in the pathogenesis of Alzheimer’s disease (AD). Thus, several therapeutic modalities aiming to inhibit Aβ synthesis or increase the clearance of Aβ have entered clinical trials, including γ-secretase inhibitors, anti-Aβ antibodies, and amyloid-β precursor protein cleaving enzyme inhibitors. A unique class of small molecules, γ-secretase modulators (GSMs), selectively reduce Aβ1-42 production, and may also decrease Aβ1-40 while simultaneously increasing one or more shorter Aβ peptides, such as Aβ1-38 and Aβ1-37. GSMs are particularly attractive because they do not alter the total amount of Aβ peptides produced by γ-secretase activity; they spare the processing of other γ-secretase substrates, such as Notch; and they do not cause accumulation of the potentially toxic processing intermediate, β-C-terminal fragment. This report describes the translation of pharmacological activity across species for two novel GSMs, (S)-7-(4-fluorophenyl)-N2-(3-methoxy-4-(3-methyl-1H-1,2,4-triazol-1-yl)phenyl)-N4-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine-2,4-diamine (BMS-932481) and (S,Z)-17-(4-chloro-2-fluorophenyl)-34-(3-methyl-1H-1,2,4-triazol-1-yl)-16,17-dihydro-15H-4-oxa-2,9-diaza-1(2,4)-cyclopenta[d]pyrimidina-3(1,3)-benzenacyclononaphan-6-ene (BMS-986133). These GSMs are highly potent in vitro, exhibit dose- and time-dependent activity in vivo, and have consistent levels of pharmacological effect across rats, dogs, monkeys, and human subjects. In rats, the two GSMs exhibit similar pharmacokinetics/pharmacodynamics between the brain and cerebrospinal fluid. In all species, GSM treatment decreased Aβ1-42 and Aβ1-40 levels while increasing Aβ1-38 and Aβ1-37 by a corresponding amount. Thus, the GSM mechanism and central activity translate across preclinical species and humans, thereby validating this therapeutic modality for potential utility in AD.

CSF neurofilament concentration reflects disease severity in frontotemporal degeneration

Cerebrospinal fluid (CSF) neurofilament light chain (NfL) concentration is elevated in neurological disorders, including frontotemporal degeneration (FTD). We investigated the clinical correlates of elevated CSF NfL levels in FTD.

Addressing drug effects on cut point determination for an anti-drug antibody assay.

The effect of trough levels of a monoclonal antibody drug (drugB) on screening cut point (CP) determination for an anti-drug antibody (ADA) assay was scrutinized and the conclusions substantiated by data from a phase 3 cancer clinical study. The ADA assay utilized an acid dissociation step and either 0 or 100 μg/ml drugB was added to the samples prior to obtaining the signals used for CP calculations. Serum samples from three different drug-naive populations were tested (healthy individuals, cancer patients enrolled in the drugB clinical trial and cancer patients whose serum samples were available commercially). For the same disease state samples, both the screening CP and confirmation CP were different when calculated during validation or from study sample analysis. It is reasonable to assume that variability was due to the patient heterogeneity, as they could have been at distinct stages of disease progression, and/or taking different medications, amongst other differences. The patients enrolled in the clinical trial were stratified as per protocol and hence represented a more homogeneous population. Drug effects on CP may be population dependent and also assay dependent.

The Gamma Secretase Inhibitor, BMS-708163 Increases Alpha Secretase Abeta Peptide Cleavage Fragments and Decreases the Gamma Secretase Abeta Peptide 1-34 Fragment in Cerebrospinal Fluid

to investigate the role of the innate immune system inAlzheimer’s disease (AD) bymeasuring complement factors in cerebrospinal fluid (CSF) of subjects with mild cognitive impairment (MCI), AD and other dementias (oDem).Methods: We selected healthy controls (n1⁄4 19, age1⁄4 63, MMSE1⁄4 29.1), subjects with MCI (n 1⁄4 56, age 1⁄4 70, MMSE 1⁄4 26.9), AD (n 1⁄4 45, age 1⁄4 72, MMSE 1⁄4 22.9) and oDem (n 1⁄4 31, age 1⁄4 69 MMSE 1⁄4 24.1) from the EDAR study (www.edarstudy.eu). We measured C3a, central to the classical, alternative and lectin complement pathways, C5a involved in the lytic complement pathway, and the soluble C5b-9 terminal complement complex (TCC). We also measured in CSF abeta 1-42, total tau, and serum amyloid P (SAP, an acute response protein which activates complement). We compared groups and correlated complement markers with other biomarkers, neuropsychological markers, andmarkers of functional impairment using non-parametric tests.Results: Complement data are shown in the table. C3a was decreased by 50-60% in subjects with MCI, AD, and oDem relative to controls (p < 0.003 all comparisons). C5a was increased 3-6 fold in AD relative to controls (p 1⁄4 0.077), MCI (p1⁄4 0.016), and oDem(p1⁄4 0.07). TCCdid not differ between the groups, although subjects with AD had higher concentrations than the other groups. In the combined sample of subjects with MCI, AD, and oDem, higher levels of C3a correlated with higher levels of TCC (r 1⁄4 0.51, p < 0.001), SAP (r 1⁄4 0.40, p< 0.001), and tau (r1⁄4 0.18, p1⁄4 0.048). Higher levels of C5a correlated with more impairment on memory, executive functioning, and the MMSE (r1⁄4 0.22-0.30, p 1⁄4 0.05-0.002). Higher levels of TCC correlated with higher SAP levels (r 1⁄4 0.41, p < 0.001) and more functional impairment (r 1⁄4 0.25, p 1⁄4 0.018).Conclusions: The innate immune system is actively involved in neurodegeneration and may provide diagnostic and prognostic information for AD. The large reduction of C3a in subjects with MCI, AD and oDem, suggests increased use of this factor, regardless of the cause of neurodegeneration. Alternatively, increased C3a levels may protect against neurodegeneration. The increase of C5a in AD suggests a disease specific activation of the lytic complement pathway. Since this measure correlated with clinical measures of severity, C5a activation may be a relatively late event in the disease process.

Cerebrospinal fluid neurofilament concentration reflects disease severity in frontotemporal degeneration: Neurofilament in FTD

Cerebrospinal fluid (CSF) neurofilament light chain (NfL) concentration is elevated in neurological disorders, including frontotemporal degeneration (FTD). We investigated the clinical correlates of elevated CSF NfL levels in FTD.

Reproducibility of a fully automated, chemiluminescent, beta-amyloid 42 assay

upon a detailed unified SOP for the INNO-BIA AlzBio3 (RUO; Innogenetics nv, Belgium) multiplexing xMAP assay. Using the same assay batch, a set of ten CSF pool samples and two buffer based control samples were analyzed using ready-to-use calibrators. A fresh aliquot of each sample was tested in quadruplicate in three independent assay runs (only 2 runs for center C). The goal of the study was to determine the with-in and interlaboratory variability of the assay for hTau, Ab 1-42, and P-Tau 181P after implementation of the unified SOP. Results: The CSF analyte concentrations showed a strong correlation between all centers (R> 0.95; regression center versus overall center mean). For the CSF samples, the total inter-center variability over the 8 runs performed (%CV) ranged between 12.4 22.9% (hTau), 8.1 13.1% (Ab 1-42), and 6.8 13.2% (P-Tau 181P). Across the 10 CSF samples, the mean within-laboratory variation ranged between 1.8% and 11% for all analytes. The variability observed for the control samples was even lower (max. 4.5%).Conclusions: Implementation of a unified SOP in the three laboratories resulted in an acceptable inter-laboratory variation of analyte concentrations for CSF and control samples. Careful documentation of the critical parameters of the test procedure and rigorous adherence to a detailed instruction of use clearly leads to highly comparable CSF analyte concentrations between experienced centers.