A Phase 1 First-in-Human Single-Ascending-Dose Trial With ESB1609, a Selective Agonist to the Sphingosine-1-Phosphate Receptor 5.

ESB1609 is a small-molecule sphingosine-1-phosphate-5 receptor-selective agonist designed to restore lipid homeostasis by promoting cytosolic egress of sphingosine-1-phosphate to reduce abnormal levels of ceramide and cholesterol in disease. A phase 1 study was conducted in healthy volunteers to determine the safety, tolerability, and pharmacokinetics of ESB1609. Following single oral doses, ESB1609 demonstrated linear pharmacokinetics in plasma and cerebrospinal fluid (CSF) for formulations containing sodium laurel sulfate. Plasma and CSF median time to maximum drug concentration (tmax ) were reached by 4-5 hours and 6-10 hours, respectively. The delay in achieving tmax in CSF relative to plasma, likely due to the high protein binding of ESB1609, was also observed in 2 rat studies. Continuous CSF collection via indwelling catheters confirmed that a highly protein-bound compound is measurable and established the kinetics of ESB1609 in human CSF. Mean plasma terminal elimination half-lives ranged from 20.2 to 26.8 hours. The effect of either a high-fat or standard meal increased maximum plasma concentration and area under the concentration-time curve from time 0 to infinity compared to the fasted state by 2.42-4.34-fold higher, but tmax and half-life remained the same irrespective of fed state. ESB1609 crosses the blood-brain barrier with CSF:plasma ratios ranging between 0.04% and 0.07% across dose levels. ESB1609 demonstrated a favorable safety and tolerability profile at exposures expected to be efficacious.

Safety and Target Engagement of Complement C1q Inhibitor ANX007 in Neurodegenerative Eye Disease: Results from Phase I Studies in Glaucoma.

Purpose: Complement C1q, the initiating molecule of the classical complement cascade, is involved in synapse elimination and neuronal loss in neurodegenerative diseases including glaucoma. Here we report an evaluation of the safety, tolerability, and ocular pharmacokinetics (PK) and pharmacodynamics of intravitreal (IVT) injections of ANX007, an anti-C1q monoclonal antibody fragment that blocks activation of the classical complement cascade. Design: An open-label, single-dose-escalation phase Ia study followed by a double-masked, randomized, sham-controlled, repeat-injection phase Ib study. Participants: A total of 26 patients with primary open-angle glaucoma. Methods: Nine patients with primary open-angle glaucoma (mean Humphrey visual field deviation between -3 and -18 decibels [dB]) were enrolled in phase Ia and received single doses of ANX007 (1.0 mg, n = 3; 2.5 mg, n = 3; or 5.0 mg, n = 3). Seventeen patients (mean Humphrey visual field deviation between -3 and -24 dB) were enrolled in phase Ib and randomized to 2 monthly doses of ANX007 (sham, n = 6; 2.5 mg ANX007, n = 6; or 5 mg ANX007, n = 5). Main Outcome Measures: Safety and tolerability (including laboratory evaluation of urinalysis, complete blood count, and serum chemistries), ANX007 PK, target engagement, and immunogenicity. Results: The mean age overall was 70 years in phase Ia and 68 years in phase Ib. In both studies, no serious adverse events were observed, no non-ocular treatment-emergent adverse events (TEAEs) attributable to study drug were reported, and ocular TEAEs were mild. Intraocular pressure returned to normal levels for all patients within 45 minutes of IVT injection. No clinically significant deviations in laboratory results were observed. In the phase Ib study, C1q in the aqueous humor was reduced to undetectable levels in both the 2.5 mg and 5 mg cohorts 4 weeks after the first ANX007 dose. Conclusions: In these studies, single and repeat IVT ANX007 injections were well tolerated and demonstrated full target engagement 4 weeks after dosing with both low and high doses, supporting monthly or less-frequent dosing. Further investigation in neurodegenerative ocular diseases is warranted. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Pharmacokinetic and Target Engagement Measures of ANX007, an Anti-C1q Antibody Fragment, Following Intravitreal Administration in Nonhuman Primates.

Purpose: C1q and the classical complement cascade are key regulators of synaptic pruning, and their aberrant activation has been implicated in neurodegenerative ophthalmic diseases including geographic atrophy and glaucoma. The antigen-binding fragment antibody ANX007 specifically recognizes globular head groups of C1q to block substrate binding and functionally inhibit classical complement cascade activation. ANX007 was assessed in nonclinical studies of biodistribution and C1q target engagement in the eye following intravitreal (IVT) administration in cynomolgus monkeys. Methods: Female juvenile cynomolgus monkeys (n = 12) received a single bilateral dose of 1 or 5 mg ANX007/eye, with vitreous and non-perfused tissue samples collected approximately 4 weeks later. In a separate study, male (n = 6/5) and female (n = 6/5) animals received repeat bilateral dosing of 1, 2.5, or 5 mg ANX007/eye on days 1 and 29, with aqueous and vitreous collections on day 44 or day 59. Tissues from the 5 mg/eye repeat-dose group were perfused, and retina, choroid, and optic nerve samples were collected approximately 2 and 4 weeks post-last dose. Results: Following a single dose of ANX007, vitreous levels of free drug were measurable through 4 weeks at both the 1 and 5 mg dose levels, with approximately 3-day half-life. With repeat dose of 5 mg/eye, free-ANX007 was measurable 4 weeks post-last dose in perfused retina and choroid and up to approximately 2 weeks post-last dose in optic nerve. There was a strong correlation between C1q target engagement and free drug levels in aqueous and vitreous humors and retinal tissue. Conclusions: Following IVT administration, ANX007 distributes to sites within the retina that are relevant to neurodegenerative ophthalmic disease with clear evidence of C1q target engagement. Based on its mechanism of action inhibiting C1q and its downstream activity, ANX007 is predicted to mitigate tissue damage driven by classical complement activation in the retina. These data support further clinical evaluation of ANX007.

Complement factor C1q mediates sleep spindle loss and epileptic spikes after mild brain injury.

Although traumatic brain injury (TBI) acutely disrupts the cortex, most TBI-related disabilities reflect secondary injuries that accrue over time. The thalamus is a likely site of secondary damage because of its reciprocal connections with the cortex. Using a mouse model of mild TBI (mTBI), we found a chronic increase in C1q expression specifically in the corticothalamic system. Increased C1q expression colocalized with neuron loss and chronic inflammation and correlated with disruption in sleep spindles and emergence of epileptic activities. Blocking C1q counteracted these outcomes, suggesting that C1q is a disease modifier in mTBI. Single-nucleus RNA sequencing demonstrated that microglia are a source of thalamic C1q. The corticothalamic circuit could thus be a new target for treating TBI-related disabilities.

The Classical Complement Pathway Mediates Microglia-Dependent Remodeling of Spinal Motor Circuits during Development and in SMA.

Movement is an essential behavior requiring the assembly and refinement of spinal motor circuits. However, the mechanisms responsible for circuit refinement and synapse maintenance are poorly understood. Similarly, the molecular mechanisms by which gene mutations cause dysfunction and elimination of synapses in neurodegenerative diseases that occur during development are unknown. Here, we demonstrate that the complement protein C1q is required for the refinement of sensory-motor circuits during normal development, as well as for synaptic dysfunction and elimination in spinal muscular atrophy (SMA). C1q tags vulnerable SMA synapses, which triggers activation of the classical complement pathway leading to microglia-mediated elimination. Pharmacological inhibition of C1q or depletion of microglia rescues the number and function of synapses, conferring significant behavioral benefit in SMA mice. Thus, the classical complement pathway plays critical roles in the refinement of developing motor circuits, while its aberrant activation contributes to motor neuron disease.

Subretinal macrophages produce classical complement activator C1q leading to the progression of focal retinal degeneration.

BACKGROUND: The role of the alternative complement pathway and its mediation by retinal microglia and macrophages, is well-established in the pathogenesis of Age-Related Macular Degeneration (AMD). However, the contribution of the classical complement pathway towards the progression of retinal degenerations is not fully understood, including the role of complement component 1q (C1q) as a critical activator molecule of the classical pathway. Here, we investigated the contribution of C1q to progressive photoreceptor loss and neuroinflammation in retinal degenerations. METHODS: Wild-type (WT), C1qa knockout (C1qa-/-) and mice treated with a C1q inhibitor (ANX-M1; Annexon Biosciences), were exposed to photo-oxidative damage (PD) and were observed for progressive lesion development. Retinal function was assessed by electroretinography, followed by histological analyses to assess photoreceptor degeneration. Retinal inflammation was investigated through complement activation, macrophage recruitment and inflammasome expression using western blotting, qPCR and immunofluorescence. C1q was localised in human AMD donor retinas using immunohistochemistry. RESULTS: PD mice had increased levels of C1qa which correlated with increasing photoreceptor cell death and macrophage recruitment. C1qa-/- mice did not show any differences in photoreceptor loss or inflammation at 7 days compared to WT, however at 14 days after the onset of damage, C1qa-/- retinas displayed less photoreceptor cell death, reduced microglia/macrophage recruitment to the photoreceptor lesion, and higher visual function. C1qa-/- mice displayed reduced inflammasome and IL-1ß expression in microglia and macrophages in the degenerating retina. Retinal neutralisation of C1q, using an intravitreally-delivered anti-C1q antibody, reduced the progression of retinal degeneration following PD, while systemic delivery had no effect. Finally, retinal C1q was found to be expressed by subretinal microglia/macrophages located in the outer retina of early AMD donor eyes, and in mouse PD retinas. CONCLUSIONS: Our data implicate subretinal macrophages, C1q and the classical pathway in progressive retinal degeneration. We demonstrate a role of local C1q produced by microglia/macrophages as an instigator of inflammasome activation and inflammation. Crucially, we have shown that retinal C1q neutralisation during disease progression may slow retinal atrophy, providing a novel strategy for the treatment of complement-mediated retinal degenerations including AMD.

Probing Conformational Dynamics of Tau Protein by Hydrogen/Deuterium Exchange Mass Spectrometry.

Fibrillization of the microtubule-associated protein tau has been recognized as one of the signature pathologies of the nervous system in Alzheimer's disease, progressive supranuclear palsy, and other tauopathies. The conformational transition of tau in the fibrillization process, tau monomer to soluble aggregates to fibrils in particular, remains unclear. Here we report on the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS) in combination with other biochemical approaches, including Thioflavin S fluorescence measurements, enzyme-linked immunosorbent assay (ELISA), and Western blotting to understand the heparin-induced tau's fibrillization. HDX-MS studies including anti-tau antibody epitope mapping experiments provided molecular level details of the full-length tau's conformational dynamics and its regional solvent accessibility upon soluble aggregates formation. The results demonstrate that R3 region in the full-length tau's microtubule binding repeat region (MTBR) is stabilized in the aggregation process, leaving both N and C terminal regions to be solvent exposed in the soluble aggregates and fibrils. The findings also illustrate the practical utility of orthogonal analytical methodologies for the characterization of protein higher order structure. Graphical Abstract ᅟ.

Nonclinical Development of ANX005: A Humanized Anti-C1q Antibody for Treatment of Autoimmune and Neurodegenerative Diseases.

ANX005 is a humanized immunoglobulin G4 recombinant antibody against C1q that inhibits its function as the initiating molecule of the classical complement cascade. The safety and tolerability of ANX005 are currently being evaluated in a phase I trial in healthy volunteers ( www.clinicaltrials.gov Identifier: NCT03010046). Inhibition of C1q can be applied therapeutically in a broad spectrum of diseases, including acute antibody-mediated autoimmune disease, such as Guillain-Barré syndrome (GBS), and in chronic diseases of the central nervous system involving complement-mediated neurodegeneration, such as Alzheimer's disease (AD). To support the clinical development of ANX005, several studies were conducted to assess the pharmacology, pharmacokinetics, and potential toxicity of ANX005. ANX-M1, the murine precursor of ANX005, functionally inhibits the classical complement cascade both in vitro and in vivo, to protect against disease pathology in mouse models of GBS and AD. Toxicology studies with ANX005, itself, showed that intravenous administration once weekly for 4 weeks was well tolerated in rats and monkeys, with no treatment-related adverse findings. Serum levels of ANX005 in monkeys correlate with a reduction in free C1q levels both in the serum and in the cerebrospinal fluid. In summary, ANX005 has shown proof of concept in in vitro and in vivo nonclinical pharmacology models, with no toxicity in the 4-week repeat-dose studies in rats and monkeys. The no observed adverse effect level was 200 mg/kg/dose, which is 200-fold higher than the first-in-human starting dose of 1 mg/kg in healthy volunteers.

C1q-targeted inhibition of the classical complement pathway prevents injury in a novel mouse model of acute motor axonal neuropathy.

INTRODUCTION: Guillain-Barré syndrome (GBS) is an autoimmune disease that results in acute paralysis through inflammatory attack on peripheral nerves, and currently has limited, non-specific treatment options. The pathogenesis of the acute motor axonal neuropathy (AMAN) variant is mediated by complement-fixing anti-ganglioside antibodies that directly bind and injure the axon at sites of vulnerability such as nodes of Ranvier and nerve terminals. Consequently, the complement cascade is an attractive target to reduce disease severity. Recently, C5 complement component inhibitors that block the formation of the membrane attack complex and subsequent downstream injury have been shown to be efficacious in an in vivo anti-GQ1b antibody-mediated mouse model of the GBS variant Miller Fisher syndrome (MFS). However, since gangliosides are widely expressed in neurons and glial cells, injury in this model was not targeted exclusively to the axon and there are currently no pure mouse models for AMAN. Additionally, C5 inhibition does not prevent the production of early complement fragments such as C3a and C3b that can be deleterious via their known role in immune cell and macrophage recruitment to sites of neuronal damage. RESULTS AND CONCLUSIONS: In this study, we first developed a new in vivo transgenic mouse model of AMAN using mice that express complex gangliosides exclusively in neurons, thereby enabling specific targeting of axons with anti-ganglioside antibodies. Secondly, we have evaluated the efficacy of a novel anti-C1q antibody (M1) that blocks initiation of the classical complement cascade, in both the newly developed anti-GM1 antibody-mediated AMAN model and our established MFS model in vivo. Anti-C1q monoclonal antibody treatment attenuated complement cascade activation and deposition, reduced immune cell recruitment and axonal injury, in both mouse models of GBS, along with improvement in respiratory function. These results demonstrate that neutralising C1q function attenuates injury with a consequent neuroprotective effect in acute GBS models and promises to be a useful new target for human therapy.

Methyl-substitution of an iminohydantoin spiropiperidine ß-secretase (BACE-1) inhibitor has a profound effect on its potency.

The IC50 of a beta-secretase (BACE-1) lead compound was improved ∼200-fold from 11 µM to 55 nM through the addition of a single methyl group. Computational chemistry, small molecule NMR, and protein crystallography capabilities were used to compare the solution conformation of the ligand under varying pH conditions to its conformation when bound in the active site. Chemical modification then explored available binding pockets adjacent to the ligand. A strategically placed methyl group not only maintained the required pKa of the piperidine nitrogen and filled a small hydrophobic pocket, but more importantly, stabilized the conformation best suited for optimized binding to the receptor.

Centrally Delivered BACE1 Inhibitor Activates Microglia, and Reverses Amyloid Pathology and Cognitive Deficit in Aged Tg2576 Mice.

Multiple small-molecule inhibitors of the ß-secretase enzyme (BACE1) are under preclinical or clinical investigation for Alzheimer's disease (AD). Prior work has illustrated robust lowering of central amyloid ß (Aß) after acute administration of BACE1 inhibitors. However, very few studies have assessed the overall impact of chronically administered BACE1 inhibitors on brain amyloid burden, neuropathology, and behavioral function in aged preclinical models. We investigated the effects of a potent nonbrain-penetrant BACE1 inhibitor, delivered directly to the brain using intracerebroventricular infusion in an aged transgenic mouse model. Intracerebroventricular infusion of the BACE1 inhibitor (0.3-23.5 µg/d) for 8 weeks, initiated in 17-month-old Tg2576 mice, produced dose-dependent increases in brain inhibitor concentrations (0.2-13 µm). BACE1 inhibition significantly reversed the behavioral deficit in contextual fear conditioning, and reduced brain Aß levels, plaque burden, and associated pathology (e.g., dystrophic neurites), with maximal effects attained with ∼1 µg/d dose. Strikingly, the BACE1 inhibitor also reversed amyloid pathology below baseline levels (amyloid burden at the start of treatment), without adversely affecting cerebral amyloid angiopathy, microhemorrhages, myelination, or neuromuscular function. Inhibitor-mediated decline in brain amyloid pathology was associated with an increase in microglial ramification. This is the first demonstration of chronically administered BACE1 inhibitor to activate microglia, reverse brain amyloid pathology, and elicit functional improvement in an aged transgenic mouse model. Thus, engagement of novel glial-mediated clearance mechanisms may drive disease-modifying therapeutic benefit with BACE1 inhibition in AD.

Passive immunization with phospho-tau antibodies reduces tau pathology and functional deficits in two distinct mouse tauopathy models.

In Alzheimer's disease (AD), an extensive accumulation of extracellular amyloid plaques and intraneuronal tau tangles, along with neuronal loss, is evident in distinct brain regions. Staging of tau pathology by postmortem analysis of AD subjects suggests a sequence of initiation and subsequent spread of neurofibrillary tau tangles along defined brain anatomical pathways. Further, the severity of cognitive deficits correlates with the degree and extent of tau pathology. In this study, we demonstrate that phospho-tau (p-tau) antibodies, PHF6 and PHF13, can prevent the induction of tau pathology in primary neuron cultures. The impact of passive immunotherapy on the formation and spread of tau pathology, as well as functional deficits, was subsequently evaluated with these antibodies in two distinct transgenic mouse tauopathy models. The rTg4510 transgenic mouse is characterized by inducible over-expression of P301L mutant tau, and exhibits robust age-dependent brain tau pathology. Systemic treatment with PHF6 and PHF13 from 3 to 6 months of age led to a significant decline in brain and CSF p-tau levels. In a second model, injection of preformed tau fibrils (PFFs) comprised of recombinant tau protein encompassing the microtubule-repeat domains into the cortex and hippocampus of young P301S mutant tau over-expressing mice (PS19) led to robust tau pathology on the ipsilateral side with evidence of spread to distant sites, including the contralateral hippocampus and bilateral entorhinal cortex 4 weeks post-injection. Systemic treatment with PHF13 led to a significant decline in the spread of tau pathology in this model. The reduction in tau species after p-tau antibody treatment was associated with an improvement in novel-object recognition memory test in both models. These studies provide evidence supporting the use of tau immunotherapy as a potential treatment option for AD and other tauopathies.

Divergent CSF τ alterations in two common tauopathies: Alzheimer's disease and progressive supranuclear palsy.

BACKGROUND: Elevated CSF τ is considered a biomarker of neuronal injury in newly developed Alzheimer's disease (AD) and mild cognitive impairment (MCI) criteria. However, previous studies have failed to detect alterations of τ species in other primary tauopathies. We assessed CSF τ protein abnormalities in AD, a tauopathy with prominent Aß pathology, and progressive supranuclear palsy (PSP), a primary tauopathy characterised by deposition of four microtubule-binding repeat (4R) τ with minimal Aß pathology. METHODS: 26 normal control (NC), 37 AD, and 24 patients with PSP participated in the study. AD and PSP were matched for severity using the clinical dementia rating sum of boxes (CDR-sb) scores. The INNO BIA AlzBio3 multiplex immunoassay was used to measure CSF Aß, total τ, and ptau181. Additional, novel ELISAs targeting different N-terminal and central τ epitopes were developed to examine CSF τ components and to investigate interactions between diagnostic group, demographics and genetic variables. RESULTS: PSP had lower CSF N-terminal and C-terminal τ concentrations than NC and AD measured with the novel τ ELISAs and the standard AlzBio3 τ and ptau assays. AD had higher total τ and ptau levels than NC and PSP. There was a gender by diagnosis interaction in AD and PSP for most τ species, with lower concentrations for male compared to female patients. CONCLUSIONS: CSF τ fragment concentrations are different in PSP compared with AD despite the presence of severe τ pathology and neuronal injury in both disorders. CSF τ concentration likely reflects multiple factors in addition to the degree of neuronal injury.

Tau overexpression impacts a neuroinflammation gene expression network perturbed in Alzheimer's disease.

Filamentous inclusions of the microtubule-associated protein, tau, define a variety of neurodegenerative diseases known as tauopathies, including Alzheimer's disease (AD). To better understand the role of tau-mediated effects on pathophysiology and global central nervous system function, we extensively characterized gene expression, pathology and behavior of the rTg4510 mouse model, which overexpresses a mutant form of human tau that causes Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We found that the most predominantly altered gene expression pathways in rTg4510 mice were in inflammatory processes. These results closely matched the causal immune function and microglial gene-regulatory network recently identified in AD. We identified additional gene expression changes by laser microdissecting specific regions of the hippocampus, which highlighted alterations in neuronal network activity. Expression of inflammatory genes and markers of neuronal activity changed as a function of age in rTg4510 mice and coincided with behavioral deficits. Inflammatory changes were tau-dependent, as they were reversed by suppression of the tau transgene. Our results suggest that the alterations in microglial phenotypes that appear to contribute to the pathogenesis of Alzheimer's disease may be driven by tau dysfunction, in addition to the direct effects of beta-amyloid.

Characterization of novel CSF Tau and ptau biomarkers for Alzheimer's disease.

Cerebral spinal fluid (CSF) Aß42, 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.

Effects of sub-chronic donepezil on brain Abeta and cognition in a mouse model of Alzheimer's disease.

RATIONALE: Acetylcholinesterase inhibitors (AChEIs) are approved to treat the symptoms of mild to moderate Alzheimer's disease by restoring acetylcholine levels at synapses where the neurotransmitter has been depleted due to neurodegeneration. This assumption is challenged by more recent clinical studies suggesting the potential for disease-modifying effects of AChEIs as well as in vitro studies showing neuroprotective effects. However, few preclinical studies have assessed whether the improvement of cognitive symptoms may be mediated by reductions in Abeta or Tau pathology. OBJECTIVES: The objective of the present study was to determine whether short-duration treatment with donepezil could improve spatial learning and memory in transgenic mice overexpressing mutant human amyloid precursor protein (hAPP) and presenilin 1 (PS1) (Dewachter et al., J Neurosci 20(17):6452-6458, 2000) after amyloid pathology has fully developed, consistent with early stages of Alzheimer'sdisease in humans. In parallel, the effect of donepezil treatment on brain amyloid, Tau, and glial endpoints was measured. RESULTS: This study showed a significant improvement in reference memory in hAPP/PS1 mice along with dose-dependent reductions in brain amyloid-ß (Aß). CONCLUSION: These results suggest that the observed cognitive improvement produced by donepezil in Alzheimer's disease may be due, at least in part, to reduction of brain Aß.

Pharmacodynamics of selective inhibition of γ-secretase by avagacestat.

A hallmark of Alzheimer's disease (AD) pathology is the accumulation of brain amyloid ß-peptide (Aß), generated by γ-secretase-mediated cleavage of the amyloid precursor protein (APP). Therefore, γ-secretase inhibitors (GSIs) may lower brain Aß 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 Aß 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) Aß 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 Aß40 and Aß42 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 Aß40. Consistent with the 137-fold selectivity measured in cell culture, we identified doses of avagacestat that reduce CSF Aß 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.

Characterization of plasma ß-secretase (BACE1) activity and soluble amyloid precursor proteins as potential biomarkers for Alzheimer's disease.

Reduction in cerebrospinal fluid (CSF) amyloid ß42 (Aß42) and elevation in total tau and phospho-thr181 tau consistently differentiate between Alzheimer's disease (AD) and age-matched control subjects. In contrast, CSF ß-site APP-cleaving enzyme activity (BACE1) and soluble amyloid precursor proteins α and ß (sAPPα and sAPPß) are without consistent patterns in AD subjects. Plasma sampling is much easier, with fewer side effects, and is readily applied in primary care centers, so we have developed and validated novel plasma BACE activity, sAPPß, and sAPPα assays and investigated their ability to distinguish AD from age-matched controls. Plasma BACE activity assay was sensitive and specific, with signal being immunodepleted with a specific BACE1 antibody and inhibited with a BACE1-specific inhibitor. Plasma sAPPß and sAPPα assays were specific, with signal diluting linearly, immunodepleted with specific antibodies, and at background levels in APP knockout mice. In rhesus monkeys, BACE1 but not γ-secretase inhibitor led to significant lowering of plasma sAPPß with concurrent elevation of plasma sAPPα. AD subjects showed a significant increase in plasma BACE1 activity, sAPPß, sAPPα, and Aß42 (P < 0.001) compared with age-matched controls. In conclusion, plasma BACE activity and sAPP endpoints provide novel investigative biomarkers for AD diagnosis and potential pharmacodynamic biomarkers for secretase inhibitor studies.

Hyperdynamic microtubules, cognitive deficits, and pathology are improved in tau transgenic mice with low doses of the microtubule-stabilizing agent BMS-241027.

Tau is a microtubule (MT)-stabilizing protein that is altered in Alzheimer's disease (AD) and other tauopathies. It is hypothesized that the hyperphosphorylated, conformationally altered, and multimeric forms of tau lead to a disruption of MT stability; however, direct evidence is lacking in vivo. In this study, an in vivo stable isotope-mass spectrometric technique was used to measure the turnover, or dynamicity, of MTs in brains of living animals. We demonstrated an age-dependent increase in MT dynamics in two different tau transgenic mouse models, 3xTg and rTg4510. MT hyperdynamicity was dependent on tau expression, since a reduction of transgene expression with doxycycline reversed the MT changes. Treatment of rTg4510 mice with the epothilone, BMS-241027, also restored MT dynamics to baseline levels. In addition, MT stabilization with BMS-241027 had beneficial effects on Morris water maze deficits, tau pathology, and neurodegeneration. Interestingly, pathological and functional benefits of BMS-241027 were observed at doses that only partially reversed MT hyperdynamicity. Together, these data suggest that tau-mediated loss of MT stability may contribute to disease progression and that very low doses of BMS-241027 may be useful in the treatment of AD and other tauopathies.

Cisterna magna cannulated repeated CSF sampling rat model--effects of a gamma-secretase inhibitor on Aß levels.

Cerebrospinal fluid (CSF) provides a window into central nervous system (CNS) physiology and pathophysiology in human neurodegenerative conditions such as Alzheimer's disease. Changes in CSF bioanalytes also provide a direct readout of target engagement in the CNS following pharmacological interventions in clinical trials. Given the importance of tracking CNS bioanalytes in drug discovery, we have developed a novel cisterna magna cannulated rat model for repeated CSF sampling and used it to assess an amyloid beta (Aß) lowering agent. The surgically implanted cisterna magna cannula was patent over a period of 1-2 weeks and enabled repeated sampling of CSF (volume of ∼30-50µL/sample) from each rat. CSF Aß40 levels showed good intra-animal variability across time points and inter-animal variability within a time point. Peripheral treatment with a gamma-secretase inhibitor (GSI) led to a rapid and robust decline in CSF Aß40 levels that returned to baseline over 24-96h after dosing. Terminal brain, CSF and plasma Aß levels measured at 24h after dosing demonstrated robust Aß lowering and showed excellent correlation across these compartments. These results are the first pharmacological validation of the repeated CSF sampling rat model for Aß lowering agents. This model can have broad applicability in pharmacological evaluation for diverse CNS targets.