CSF biomarker concordance with amyloid PET in Phase 3 studies of aducanumab.

INTRODUCTION: We assessed the use of cerebrospinal fluid (CSF) biomarkers as an alternative to positron emission tomography (PET) for brain amyloid beta (Aß) pathology confirmation in the EMERGE and ENGAGE clinical trials. METHODS: EMERGE and ENGAGE were randomized, placebo-controlled, Phase 3 trials of aducanumab in participants with early Alzheimer's disease. Concordance between CSF biomarkers (Aß42, Aß40, phosphorylated tau 181, and total tau) and amyloid PET status (visual read) at screening was examined. RESULTS: Robust concordance between CSF biomarkers and amyloid PET visual status was observed (for Aß42/Aß40, AUC: 0.90; 95% CI: 0.83-0.97; p < 0.0001), confirming CSF biomarkers as a reliable alternative to amyloid PET in these studies. Compared with single CSF biomarkers, CSF biomarker ratios showed better agreement with amyloid PET visual reads, demonstrating high diagnostic accuracy. DISCUSSION: These analyses add to the growing body of evidence supporting CSF biomarkers as reliable alternatives to amyloid PET imaging for brain Aß pathology confirmation. HIGHLIGHTS: CSF biomarkers and amyloid PET concordance were assessed in Ph3 aducanumab trials. Robust concordance between CSF biomarkers and amyloid PET was observed. CSF biomarker ratios increased diagnostic accuracy over single CSF biomarkers. CSF Aß42/Aß40 demonstrated high concordance with amyloid PET. Results support CSF biomarker testing as a reliable alternative to amyloid PET.

Comparative analytical performance of multiple plasma Aß42 and Aß40 assays and their ability to predict positron emission tomography amyloid positivity.

INTRODUCTION: This report details the approach taken to providing a dataset allowing for analyses on the performance of recently developed assays of amyloid beta (Aß) peptides in plasma and the extent to which they improve the prediction of amyloid positivity. METHODS: Alzheimer's Disease Neuroimaging Initiative plasma samples with corresponding amyloid positron emission tomography (PET) data were run on six plasma Aß assays. Statistical tests were performed to determine whether the plasma Aß measures significantly improved the area under the receiver operating characteristic curve for predicting amyloid PET status compared to age and apolipoprotein E (APOE) genotype. RESULTS: The age and APOE genotype model predicted amyloid status with an area under the curve (AUC) of 0.75. Three assays improved AUCs to 0.81, 0.81, and 0.84 (P < .05, uncorrected for multiple comparisons). DISCUSSION: Measurement of Aß in plasma contributes to addressing the amyloid component of the ATN (amyloid/tau/neurodegeneration) framework and could be a first step before or in place of a PET or cerebrospinal fluid screening study. HIGHLIGHTS: The Foundation of the National Institutes of Health Biomarkers Consortium evaluated six plasma amyloid beta (Aß) assays using Alzheimer's Disease Neuroimaging Initiative samples. Three assays improved prediction of amyloid status over age and apolipoprotein E (APOE) genotype. Plasma Aß42/40 predicted amyloid positron emission tomography status better than Aß42 or Aß40 alone.

Reference measurement procedures for Alzheimer's disease cerebrospinal fluid biomarkers: definitions and approaches with focus on amyloid ß42.

Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) are increasingly used in clinical settings, research and drug trials. However, their broad-scale use on different technology platforms is hampered by the lack of standardization at the level of sample handling, determination of concentrations of analytes and the absence of well-defined performance criteria for in vitro diagnostic or companion diagnostic assays, which influences the apparent concentration of the analytes measured and the subsequent interpretation of the data. There is a need for harmonization of CSF AD biomarker assays that can reliably, across centers, quantitate CSF biomarkers with high analytical precision, selectivity and stability over long time periods. In this position paper, we discuss reference procedures for the measurement of CSF AD biomarkers, especially amyloid ß42 and tau. We describe possible technical approaches, focusing on a selected reaction monitoring mass spectrometry assay as a candidate reference method for quantification of CSF amyloid ß42.

Purinergic receptor-mediated morphological changes in microglia are transient and independent from inflammatory cytokine release.

Microglia are commonly described as existing in resting or active states based on morphology or level of cytokine production. Extracellular ATP is a physiologically-relevant activator of microglia, which express a number of purinergic receptors. As P2Y(12) has been linked to chemotaxis, we used a panel of purinergic compounds to understand the role of ATP receptors in morphological transformation and correlate this with TNFalpha production. We quantified activation of cultured microglia with LPS or purinergic receptor agonists by using automated image analysis of cell morphology and CD11b expression and correlated this with TNFalpha release measured by ELISA. Treatment with both ATP and the P2Y(12) receptor agonist, 2-methylthio adenosine diphosphate (2MeSADP), caused a transient increase in CD11b expression (EC(50)=1.2 microM and 187 nM, respectively) and a reduction in process count that reversed within 90 min later. These changes were not accompanied by the release of TNFalpha. Forskolin, IBMX, and pertussis toxin inhibited these changes, but the PLC inhibitor, U73122, did not. 2MeSAMP blocked the ATP response, while AP4A blocked the 2MeSADP response, implicating P2Y(12/13). Microglia activation by LPS also caused an increase in CD11b expression and a reduction in process count; however, in contrast to activation by ATP, morphological transformation was accompanied by a concentration-dependent increase in TNFalpha secretion These data demonstrate that morphological transformation and TNFalpha release are separable events mediated by different, or non-convergent pathways and that although ATP can initiate morphological changes, additional factors are required to maintain activation over sustained periods.

Begacestat (GSI-953): a novel, selective thiophene sulfonamide inhibitor of amyloid precursor protein gamma-secretase for the treatment of Alzheimer's disease.

The presenilin containing gamma-secretase complex is responsible for the regulated intramembraneous proteolysis of the amyloid precursor protein (APP), the Notch receptor, and a multitude of other substrates. gamma-Secretase catalyzes the final step in the generation of Abeta(40) and Abeta(42) peptides from APP. Amyloid beta-peptides (Abeta peptides) aggregate to form neurotoxic oligomers, senile plaques, and congophilic angiopathy, some of the cardinal pathologies associated with Alzheimer's disease. Although inhibition of this protease acting on APP may result in potentially therapeutic reductions of neurotoxic Abeta peptides, nonselective inhibition of the enzyme may cause severe adverse events as a result of impaired Notch receptor processing. Here, we report the preclinical pharmacological profile of GSI-953 (begacestat), a novel thiophene sulfonamide gamma-secretase inhibitor (GSI) that selectively inhibits cleavage of APP over Notch. This GSI inhibits Abeta production with low nanomolar potency in cellular and cell-free assays of gamma-secretase function, and displaces a tritiated analog of GSI-953 from enriched gamma-secretase enzyme complexes with similar potency. Cellular assays of Notch cleavage reveal that this compound is approximately 16-fold selective for the inhibition of APP cleavage. In the human APP-overexpressing Tg2576 transgenic mouse, treatment with this orally active compound results in a robust reduction in brain, plasma, and cerebral spinal fluid Abeta levels, and a reversal of contextual fear-conditioning deficits that are correlated with Abeta load. In healthy human volunteers, oral administration of a single dose of GSI-953 produces dose-dependent changes in plasma Abeta levels, confirming pharmacodynamic activity of GSI-953 in humans.

Synthesis and structure-activity relationship of a novel series of heterocyclic sulfonamide gamma-secretase inhibitors.

gamma-Secretase inhibitors have been shown to reduce the production of beta-amyloid, a component of the plaques that are found in brains of patients with Alzheimer's disease. A novel series of heterocyclic sulfonamide gamma-secretase inhibitors that reduce beta-amyloid levels in cells is reported. Several examples of compounds within this series demonstrate a higher propensity to inhibit the processing of amyloid precursor protein compared to Notch, an alternative gamma-secretase substrate.

(S)-N-(5-Chlorothiophene-2-sulfonyl)-beta,beta-diethylalaninol a Notch-1-sparing gamma-secretase inhibitor.

Accumulation of beta-amyloid (Abeta), produced by the proteolytic cleavage of amyloid precursor protein (APP) by beta- and gamma-secretase, is widely believed to be associated with Alzheimer's disease (AD). Research around the high-throughput screening hit (S)-4-chlorophenylsulfonyl isoleucinol led to the identification of the Notch-1-sparing (9.5-fold) gamma-secretase inhibitor (S)-N-(5-chlorothiophene-2-sulfonyl)-beta,beta-diethylalaninol 7.b.2 (Abeta(40/42) EC(50)=28 nM), which is efficacious in reduction of Abeta production in vivo.

Discovery of begacestat, a Notch-1-sparing gamma-secretase inhibitor for the treatment of Alzheimer's disease.

SAR on HTS hits 1 and 2 led to the potent, Notch-1-sparing GSI 9, which lowered brain Abeta in Tg2576 mice at 100 mg/kg po. Converting the metabolically labile methyl groups in 9 to trifluoromethyl groups afforded the more stable analogue 10, which had improved in vivo potency. Further side chain modification afforded the potent Notch-1-sparing GSI begacestat (5), which was selected for development for the treatment of Alzheimer's disease.

Discovery of a novel series of Notch-sparing gamma-secretase inhibitors.

Using a cell-based assay, we have identified a new series of Notch-sparing gamma-secretase inhibitors from HTS screening leads 2a and 2e. Lead optimization studies led to the discovery of analog 8e with improved gamma-secretase inhibitory potency and Notch-sparing selectivity.

Enhanced clearance of Abeta in brain by sustaining the plasmin proteolysis cascade.

The amyloid hypothesis states that a variety of neurotoxic beta-amyloid (Abeta) species contribute to the pathogenesis of Alzheimer's disease. Accordingly, a key determinant of disease onset and progression is the appropriate balance between Abeta production and clearance. Enzymes responsible for the degradation of Abeta are not well understood, and, thus far, it has not been possible to enhance Abeta catabolism by pharmacological manipulation. We provide evidence that Abeta catabolism is increased after inhibition of plasminogen activator inhibitor-1 (PAI-1) and may constitute a viable therapeutic approach for lowering brain Abeta levels. PAI-1 inhibits the activity of tissue plasminogen activator (tPA), an enzyme that cleaves plasminogen to generate plasmin, a protease that degrades Abeta oligomers and monomers. Because tPA, plasminogen and PAI-1 are expressed in the brain, we tested the hypothesis that inhibitors of PAI-1 will enhance the proteolytic clearance of brain Abeta. Our data demonstrate that PAI-1 inhibitors augment the activity of tPA and plasmin in hippocampus, significantly lower plasma and brain Abeta levels, restore long-term potentiation deficits in hippocampal slices from transgenic Abeta-producing mice, and reverse cognitive deficits in these mice.

The LXR agonist TO901317 selectively lowers hippocampal Abeta42 and improves memory in the Tg2576 mouse model of Alzheimer's disease.

Recent studies show that intracellular cholesterol levels can modulate the processing of amyloid precursor protein to Abeta peptide. Moreover, cholesterol-rich apoE-containing lipoproteins may also promote Abeta clearance. Agonists of the liver X receptor (LXR) transcriptionally induce genes involved in intracellular lipid efflux and transport, including apoE. Thus, LXR agonists have the potential to both inhibit APP processing and promote Abeta clearance. Here we show that LXR agonist, TO901317, increased hippocampal ABCA1 and apoE and decreased Abeta42 levels in APP transgenic mice. TO901317 had no significant effects on levels of Abeta40, full length APP, or the APP processing products. Next, we examined the effects of TO901317 in the contextual fear conditioning paradigm; TO901317 completely reversed the contextual memory deficit in these mice. These data demonstrate that LXR agonists do not directly inhibit APP processing but rather facilitate the clearance of Abeta42 and may represent a novel therapeutic approach to Alzheimer's disease.

Early-onset behavioral and synaptic deficits in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder for which numerous mouse models have been generated. In both AD patients and mouse models, there is increasing evidence that neuronal dysfunction occurs before the accumulation of beta-amyloid (Abeta)-containing plaques and neurodegeneration. Characterization of the timing and nature of preplaque dysfunction is important for understanding the progression of this disease and to identify pathways and molecular targets for therapeutic intervention. Hence, we have examined the progression of dysfunction at the morphological, functional, and behavioral levels in the Tg2576 mouse model of AD. Our data show that decreased dendritic spine density, impaired long-term potentiation (LTP), and behavioral deficits occurred months before plaque deposition, which was first detectable at 18 months of age. We detected a decrease in spine density in the outer molecular layer of the dentate gyrus (DG) beginning as early as 4 months of age. Furthermore, by 5 months, there was a decline in LTP in the DG after perforant path stimulation and impairment in contextual fear conditioning. Moreover, an increase in the Abeta42/Abeta40 ratio was first observed at these early ages. However, total amyloid levels did not significantly increase until approximately 18 months of age, at which time significant increases in reactive astrocytes and microglia could be observed. Overall, these data show that the perforant path input from the entorhinal cortex to the DG is compromised both structurally and functionally, and this pathology is manifested in memory defects long before significant plaque deposition.

Acute gamma-secretase inhibition improves contextual fear conditioning in the Tg2576 mouse model of Alzheimer's disease.

Transgenic mice (Tg2576) overexpressing the Swedish mutation of the human amyloid precursor protein display biochemical, pathological, and behavioral markers consistent with many aspects of Alzheimer's disease, including impaired hippocampal function. Impaired, hippocampal-dependent, contextual fear conditioning (CFC) is observed in mice as young as 20 weeks of age. This impairment can be attenuated after treatment before training with the phosphodiesterase-4 inhibitor rolipram (0.1 mg/kg, i.p.). A rolipram-associated improvement is also observed in the littermate controls, suggesting that the effect of rolipram is independent of beta-amyloid. Acute treatment before training (but not after training or before testing) with the gamma-secretase inhibitor (GSI) N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine-t-butylester (DAPT), at a dose that reduces brain concentrations of beta-amyloid (100 mg/kg), attenuates the impairment in 20- to 65-week-old Tg2576 mice. Importantly, DAPT had no effect on performance of control littermates. These data are supportive of a role of beta-amyloid in the impairment of CFC in Tg2576 mice. Furthermore, they suggest that acute treatment with GSI may provide improved cognitive functioning as well as disease-modifying effects in Alzheimer's disease.

Molecular-modeling based design, synthesis, and activity of substituted piperidines as gamma-secretase inhibitors.

Alzheimer's disease (AD) is a debilitating disease widely thought to be associated with the accumulation of beta amyloid (Abeta) in the brain. Inhibition of gamma-secretase, one of the enzymes responsible for Abeta production, may be a useful strategy for the treatment of AD. Described below is a series of gamma-secretase inhibitors designed from a scaffold identified by a ROCS [J. Comput. Chem.1996, 17, 1653] search of the corporate database.