Paul Rhyne

Pharmacodynamics of Selective Inhibition of γ -Secretase by Avagacestat

A hallmark of Alzheimer’s disease (AD) pathology is the accumulation of brain amyloid β-peptide (Aβ), generated by γ-secretase-mediated cleavage of the amyloid precursor protein (APP). Therefore, γ-secretase inhibitors (GSIs) may lower brain Aβ and offer a potential new approach to treat AD. As γ-secretase also cleaves Notch proteins, GSIs can have undesirable effects due to interference with Notch signaling. Avagacestat (BMS-708163) is a GSI developed for selective inhibition of APP over Notch cleavage. Avagacestat inhibition of APP and Notch cleavage was evaluated in cell culture by measuring levels of Aβ and human Notch proteins. In rats, dogs, and humans, selectivity was evaluated by measuring plasma blood concentrations in relation to effects on cerebrospinal fluid (CSF) Aβ levels and Notch-related toxicities. Measurements of Notch-related toxicity included goblet cell metaplasia in the gut, marginal-zone depletion in the spleen, reductions in B cells, and changes in expression of the Notch-regulated hairy and enhancer of split homolog-1 from blood cells. In rats and dogs, acute administration of avagacestat robustly reduced CSF Aβ40 and Aβ42 levels similarly. Chronic administration in rats and dogs, and 28-day, single- and multiple-ascending–dose administration in healthy human subjects caused similar exposure-dependent reductions in CSF Aβ40. Consistent with the 137-fold selectivity measured in cell culture, we identified doses of avagacestat that reduce CSF Aβ levels without causing Notch-related toxicities. Our results demonstrate the selectivity of avagacestat for APP over Notch cleavage, supporting further evaluation of avagacestat for AD therapy.

Recommendations for adaptation and validation of commercial kits for biomarker quantification in drug development

Increasingly, commercial immunoassay kits are used to support drug discovery and development. Longitudinally consistent kit performance is crucial, but the degree to which kits and reagents are characterized by manufacturers is not standardized, nor are the approaches by users to adapt them and evaluate their performance through validation prior to use. These factors can negatively impact data quality. This paper offers a systematic approach to assessment, method adaptation and validation of commercial immunoassay kits for quantification of biomarkers in drug development, expanding upon previous publications and guidance. These recommendations aim to standardize and harmonize user practices, contributing to reliable biomarker data from commercial immunoassays, thus, enabling properly informed decisions during drug development.

Electrochemiluminescence in bioanalysis

The discovery of electrochemiluminescence (ECL) and its development as a means of detection is truly a success story. Although studies describing ECL were published in the early 1960s, most studies using ECL as a means of detection were not widely published until the mid 1990s. Incorporating ECL into assays provides increased sensitivity, several logs of dynamic range and the ability to electronically control the reaction. These characteristics provide advantages over assays that rely on radioisotopic labels, fluorescence and enzymatic activity. There have been many areas of science that have benefited from the use of ECL, including environmental microbiology, virology, neurobiology, molecular biology and immunology. ECL has improved the understanding and treatment of infectious diseases, cancer, neurodegenerative diseases and even sleep apnea disorders. Drug development has also benefited from ECL via improved assessment of pharmacodynamics, pharmacokinetics and determining immune responses against protein-based therapeutics. This review provides an overview of ECL chemistry and principles with a more detailed emphasis on the applications of ECL-based assays in different areas of science and medicine. The primary purpose of this review is to provide an in-depth discussion of the impact that ECL-based analysis has had on microbiology, immunology, virology, neurodegenerative diseases, molecular biology and drug development. Examples of ECL-based bioanalysis in each of these fields are discussed in conjunction with an overview of ECL principles and instrumentation.

Immunoassay-based measurement of clinical biomarkers for monitoring changes in nasal cavity

BACKGROUND Many drugs for treatment of allergies, migraine headaches, inflammation, and other indications are administered into the nasal cavity providing access to the immune and central nervous systems. One of the concerns for using this route of administration is potential damage to the nasal epithelium and mucosal regions. We assembled a panel of clinical biomarkers that can be used to monitor changes in the nasal epithelium, mucosa, and olfactory regions in preparation for clinical trials involving drugs administered via intranasal route. These biomarkers included albumin, elastase, IL-6, IL-8, lactoferrin, myeloperoxidase and nerve growth factor. METHODS Immunoassays were developed and used to measure changes in these biomarkers in nasal lavage samples collected twice daily from 30 assumed-healthy volunteers over a 2-day period. Various statistical methods including analysis of variance (ANOVA), paired t-test and Pearsons product-moment correlation were used to evaluate the data. RESULTS Although the basal levels of these biomarkers were varied among subjects, the data show that the concentrations of albumin, elastase and IL-8 were significantly higher in samples collected in the morning compared to samples collected later during the day. Pre-washing nasal cavity prior to collecting nasal lavage samples did alter the measurement of elastase and albumin, but did not influence the levels of the other biomarkers. CONCLUSIONS These data show that this panel of biomarkers can be used to monitor changes in the nasal cavity including those affected by diurnal fluctuations. These results also provide useful baseline values and sources of variability for each biomarker that could be used to help design clinical trials.

8th GCC: Consolidated feedback to US FDA on the 2013 Draft FDA Guidance on Bioanalytical Method Validation

The 8th GCC Closed Forum for Bioanalysis was held in Baltimore, MD, USA on 5 December 2013, immediately following the 2013 AAPS Workshop (Crystal City V): Quantitative Bioanalytical Methods Validation and Implementation--The 2013 Revised FDA Guidance. This GCC meeting was organized to discuss the contents of the draft revised FDA Guidance on bioanalytical method validation that was published in September 2013 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants, from seven countries, representing 46 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the draft FDA Guidance, and to build unified comments to be provided to the FDA.

The 10th GCC Closed Forum: rejected data, GCP in bioanalysis, extract stability, BAV, processed batch acceptance, matrix stability, critical reagents, ELN and data integrity and counteracting fraud

The 10th Global CRO Council (GCC) Closed Forum was held in Orlando, FL, USA on 18 April 2016. In attendance were decision makers from international CRO member companies offering bioanalytical services. The objective of this meeting was for GCC members to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at this closed forum included reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, biomarker assay validation, processed batch acceptance criteria, electronic laboratory notebooks and data integrity, Health Canadas Notice regarding replicates in matrix stability evaluations, critical reagents and regulatory approaches to counteract fraud. In order to obtain the pharma perspectives on some of these topics, the first joint CRO-Pharma Scientific Interchange Meeting was held on 12 November 2016, in Denver, Colorado, USA. The five topics discussed at this Interchange meeting were reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, processed batch acceptance criteria and electronic laboratory notebooks and data integrity. The conclusions from the discussions of these topics at both meetings are included in this report.

Characterization and development of a Luminex®-based assay for the detection of human IL-23

BACKGROUND IL-23 is a cytokine produced by dendritic cells, T-cells and macrophages that plays a critical regulatory role in the inflammatory and autoimmune responses. We describe the development and preclinical validation of a highly sensitive Luminex(®) assay specific to IL-23 that is suitable for its measurement in support of early-phase clinical trials. RESULTS Intra-assay precision for the BioSource™ ELISA was under 12.3%, and under 5.2% for the eBioscience(®) ELISA. In comparison, the Luminex assays provided an intra-assay precision under 6.2%. The measured inter-assay precision was less than 15.6% for the BioSource ELISA, under 33% for the eBioscience and less than 10% for the Luminex assays. CONCLUSIONS The Luminex method described provides a way to measure IL-23 in clinical samples either as a single biomarker or as a panel of biomarkers. The assay should prove useful to scientists and clinicians investigating the biology of IL-23 and to those needing to monitor changes in IL-23 as part of a clinical study.

Recommendations for Selection and Characterization of Protein Biomarker Assay Calibrator Material

As biomarkers continue to become an integral part of drug development and decision-making, there are increased expectations for reliable and quantitative assays. Protein biomarker assay results are directly influenced by the calibrator material. The selection of calibrator material presents many challenges that impact the relative accuracy and performance of the assay. There is an industry-wide challenge finding reliable and well-characterized calibrator material with good documentation. Several case studies are presented that demonstrate some of the challenges involved in selecting appropriate calibrators along with the resolutions that were ultimately applied. From these experiences, we present here a set of recommendations for selecting and characterizing calibrator material based on the intended purpose of the assay. Finally, we introduce a commutability approach, based on common clinical chemistry practices, which can be used to demonstrate inter-changeability with calibrator materials across multiple lots and technology platforms for all types of protein biomarker assays.

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.

Fit for purpose Clinical validation of a multiplex immunoassay for the measurement of Tau, p-Tau and Abeta 42

Methods: For this purpose, organotypic hippocampal cultures (OHCs) were exposed to Aß1-42 (2 mM) for 48 hours with or without curcumin (0.5, 1, 5 and 10 mM). We also analysed the effect of LY294002 (5 mM), an inhibitor of phosphoinositide-3-kinase (PI3-K) pathway in OHCs exposed to Aß1-42 (2 mM) and curcumin (10 mM). Cell death was measured by propidium iodide uptake in the slices and some cell signaling pathways were investigated by performing Western blot assay with specifics antibodies. Moreover, we measured the synaptophysin expression, involved in the regulation of synaptic plasticity, by Western blot assay. Results:Our results show that Aß1-42 peptide caused about 30% of cell damage in hippocampal slices, a significant increase when compared to controls cultures. The treatment with 5 and 10 mM of curcumin decreased the cell death significantly. The curcumin treatment prevented the decreased in synaptophysin expression after exposure to Aß peptide. Aß1-42 neurotoxicity resulted in an increased in phosphorylated (Ser45) ß-catenin and a decreased in ß-catenin immunocontent and the curcumin treatment prevented this ß-catenin destabilization. Additionally, the curcumin (10 mM) neuroprotection was prevented by LY294002. Immunoblotting revealed that curcumin induced the phosphorylation/activation of Akt and the phosphorylation/inactivation of glycogen synthase kinase-3ß (GSK-3ß). Conclusions: These results reinforce the neuroprotective effect of curcumin and add some evidence that its mechanism may involve the PI3-K pathway and ß-catenin signaling, a key transducer of the Wnt signaling pathway. We also provide interesting perspectives on the use of our model in the study of this disease process.