Pre-clinical characterisation of E2814, a high-affinity antibody targeting the microtubule-binding repeat domain of tau for passive immunotherapy in Alzheimer's disease.

Tau deposition in the brain is a pathological hallmark of many neurodegenerative disorders, including Alzheimer's disease (AD). During the course of these tauopathies, tau spreads throughout the brain via synaptically-connected pathways. Such propagation of pathology is thought to be mediated by tau species ("seeds") containing the microtubule binding region (MTBR) composed of either three repeat (3R) or four repeat (4R) isoforms. The tau MTBR also forms the core of the neuropathological filaments identified in AD brain and other tauopathies. Multiple approaches are being taken to limit tau pathology, including immunotherapy with anti-tau antibodies. Given its key structural role within fibrils, specifically targetting the MTBR with a therapeutic antibody to inhibit tau seeding and aggregation may be a promising strategy to provide disease-modifying treatment for AD and other tauopathies. Therefore, a monoclonal antibody generating campaign was initiated with focus on the MTBR. Herein we describe the pre-clinical generation and characterisation of E2814, a humanised, high affinity, IgG1 antibody recognising the tau MTBR. E2814 and its murine precursor, 7G6, as revealed by epitope mapping, are antibodies bi-epitopic for 4R and mono-epitopic for 3R tau isoforms because they bind to sequence motif HVPGG. Functionally, both antibodies inhibited tau aggregation in vitro. They also immunodepleted a variety of MTBR-containing tau protein species. In an in vivo model of tau seeding and transmission, attenuation of deposition of sarkosyl-insoluble tau in brain could also be observed in response to antibody treatment. In AD brain, E2814 bound different types of tau filaments as shown by immunogold labelling and recognised pathological tau structures by immunohistochemical staining. Tau fragments containing HVPGG epitopes were also found to be elevated in AD brain compared to PSP or control. Taken together, the data reported here have led to E2814 being proposed for clinical development.

Preclinical in vivo characterization of lemborexant (E2006), a novel dual orexin receptor antagonist for sleep/wake regulation.

STUDY OBJECTIVES: To present results from in vivo studies underlying the preclinical development of lemborexant (E2006), a novel dual orexin (hypocretin) receptor antagonist for sleep/wake regulation. METHODS: Rodent (wild-type rats and wild-type and orexin neuron-deficient [orexin/ataxin-3 Tg/+] mice) studies were performed to evaluate the effects of single-dose oral lemborexant (1-300 mg/kg) on orexin-induced increases in plasma adrenocorticotropic hormone (ACTH), locomotor activity, vigilance state measures (wakefulness, nonrapid eye movement [non-REM] sleep, rapid eye movement [REM] sleep), ethanol-induced anesthesia, and motor coordination, and the effects of multiple-dose oral lemborexant (30 mg/kg) on vigilance state measures. Active comparators were almorexant and zolpidem. Pharmacokinetics were assessed after single-dose lemborexant in mice and rats. RESULTS: Lemborexant prevented the orexin-promoted increase in ACTH in rats, therefore demonstrating inhibition of the orexin signaling pathway. Furthermore, lemborexant promoted sleep in wild-type mice and rats. Lemborexant promoted REM and non-REM sleep at an equal rate (there was no change in the REM sleep ratio). In contrast, zolpidem reduced REM sleep. The sleep-promoting effect of lemborexant was mediated via the orexin-peptide signaling pathway as demonstrated by a lack of sleep promotion in orexin neuron-deficient mice. Chronic dosing was not associated with a change in effect size or sleep architecture immediately postdosing. Lemborexant did not increase the sedative effects of ethanol or impair motor coordination, showing good safety margin in animals. Pharmacokinetic/pharmacodynamic data for mice and rats were well aligned. CONCLUSIONS: These findings supported further clinical evaluation (ongoing at this time) of lemborexant as a potential candidate for treating insomnia and other sleep disorders.

PE859, a novel tau aggregation inhibitor, reduces aggregated tau and prevents onset and progression of neural dysfunction in vivo.

In tauopathies, a neural microtubule-associated protein tau (MAPT) is abnormally aggregated and forms neurofibrillary tangle. Therefore, inhibition of the tau aggregation is one of the key approaches for the treatment of these diseases. Here, we have identified a novel tau aggregation inhibitor, PE859. An oral administration of PE859 resulted in the significant reduction of sarkosyl-insoluble aggregated tau along with the prevention of onset and progression of the motor dysfunction in JNPL3 P301L-mutated human tau transgenic mice. These results suggest that PE859 is useful for the treatment of tauopathies.

Donepezil attenuates excitotoxic damage induced by membrane depolarization of cortical neurons exposed to veratridine.

Long-lasting membrane depolarization in cerebral ischemia causes neurotoxicity via increases of intracellular sodium concentration ([Na+]i) and calcium concentration ([Ca2+]i). Donepezil has been shown to exert neuroprotective effects in an oxygen-glucose deprivation model. In the present study, we examined the effect of donepezil on depolarization-induced neuronal cell injury resulting from prolonged opening of Na+ channels with veratridine in rat primary-cultured cortical neurons. Veratridine (10 microM)-induced neuronal cell damage was completely prevented by 0.1 microM tetrodotoxin. Pretreatment with donepezil (0.1-10 microM) for 1 day significantly decreased cell death in a concentration-dependent manner, and a potent NMDA receptor antagonist, dizocilpine (MK801), showed a neuroprotective effect at the concentration of 10 microM. The neuroprotective effect of donepezil was not affected by nicotinic or muscarinic acetylcholine receptor antagonists. We further characterized the neuroprotective properties of donepezil by measuring the effect on [Na+]i and [Ca2+]i in cells stimulated with veratridine. At 0.1-10 microM, donepezil significantly and concentration-dependently reduced the veratridine-induced increase of [Ca2+]i, whereas MK801 had no effect. At 10 microM, donepezil significantly decreased the veratridine-induced increase of [Na+]i. We also measured the effect on veratridine-induced release of the excitatory amino acids, glutamate and glycine. While donepezil decreased the release of glutamate and glycine, MK801 did not. In conclusion, our results indicate that donepezil has neuroprotective activity against depolarization-induced toxicity in rat cortical neurons via inhibition of the rapid influx of sodium and calcium ions, and via decrease of glutamate and glycine release, and also that this depolarization-induced toxicity is mediated by glutamate receptor activation.

Protective effect of donepezil in primary-cultured rat cortical neurons exposed to N-methyl-d-aspartate (NMDA) toxicity.

Donepezil has a neuroprotective effect against oxygen-glucose deprivation injury and glutamate toxicity in cultured cortical neurons. In this study, we further characterized the neuroprotective properties of donepezil in rat cortical cell cultures using glutamate receptor-specific agonists (N-methyl-d-aspartate (NMDA), alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) and kainate). Pretreatment with donepezil (1 microM) for 12 h significantly decreased the lactate dehydrogenase (LDH) release in response to NMDA (100 microM) by 43.8%, and reduced the LDH release in response to kainate (100 microM) and AMPA (100 microM) by 11.9% and 7.5% (without statistical significance), respectively. Donepezil appeared to inhibit LDH release in a concentration-dependent manner at 0.1-10 microM. Cortical neurons exposed to NMDA retained a normal morphological appearance in the presence of 10 microM donepezil. In binding assay for glutamate receptors, donepezil at 100 microM only slightly inhibited binding to the glycine and polyamine sites on NMDA receptor complex. On the other hand, 12 h pretreatment with donepezil at 10 and 100 microM significantly decreased the NMDA-induced increase of intracellular calcium concentration ([Ca2+]i). In conclusion, our results show that donepezil has protective activity against NMDA toxicity in cortical neurons, and this neuroprotection seems to be partially mediated by inhibition of the increase of [Ca2+]i.

Protective effect of donepezil against Abeta(1-40) neurotoxicity in rat septal neurons.

Donepezil, a potent acetylcholinesterase (AChE) inhibitor used for the treatment of Alzheimer's disease (AD), is thought to have a neuroprotective effect in AD patients. Because a deficit in cholinergic neurotransmission is a major feature in AD, and amyloid-beta (Abeta) accumulation has been proposed as a possible causative phenomenon, we were interested to examine the effect of donepezil on Abeta(1-40) induced neurotoxicity in primary cultures of rat septal neurons. Using immunohistochemical staining, almost all the neurons were found to be positive for vesicular acetylcholine transporter (VAChT) in these septal cultures. Septal neuronal cells were cultured for 7 days and then 15 micromol/L of Abeta(1-40) was added to the cell medium for 48 h. The cultured septal neurons were highly susceptible to Abeta toxicity, as shown by morphological examination and lactate dehydrogenase (LDH) assay. Donepezil concentration-dependently reduced the LDH efflux induced by Abeta(1-40), and the effect was significant at 100 nmol/L and above. Donepezil decreased both the negative peak at around 215 nm in the circular dichroism (CD) spectrum and the fluorescence intensity of thioflavin T in the presence of Abeta(1-40). These results suggest that donepezil exerts a neuroprotective effect by reducing the amount of the toxic form of Abeta fibrils in septal neuron cultures. These findings support the idea that the clinical efficacy of donepezil in AD is due to not only activation of cholinergic transmission, but also attenuation of neuronal damage.

Protective effect of donepezil in a primary culture of rat cortical neurons exposed to oxygen-glucose deprivation.

Donepezil hydrochloride (donepezil: (+/-)-2-[(1-benzylpiperidin-4-yl)methyl]-5,6-dimethoxy-indan-1-one monohydrochloride) is a potent acetylcholinesterase inhibitor used for treatment of Alzheimer's disease. Although acetylcholinesterase inhibitors are used as a symptomatic treatment for Alzheimer's disease, it is not clear whether or not they are effective against progressive degeneration of neuronal cells. In this study, we investigated the neuroprotective effects of donepezil and other acetylcholinesterase inhibitors used for treatment of Alzheimer's disease, i.e., galantamine, rivastigmine, and tacrine. As a neurodegenerative model, we used rat cortical neurons exposed to oxygen-glucose deprivation. Lactate dehydrogenase (LDH) released into the culture medium was measured as a marker of neuronal cell damage. First, the effects of donepezil (10 microM) on three different treatment schedules (from 12 h before to 24 h after oxygen-glucose deprivation (pre-12 h), from 1 h before to 24 h after oxygen-glucose deprivation (pre-1 h) and from 1 h after to 24 h after oxygen-glucose deprivation (post-1 h)) were compared. The pre-12-h treatment most effectively inhibited LDH release. The protective effect of donepezil was confirmed morphologically. Next, the effects of donepezil and the other three acetylcholinesterase inhibitors were compared under the pre-12-h treatment condition. Donepezil (0.1, 1, and 10 microM) significantly decreased LDH release in a concentration-dependent manner. However, galantamine (1, 10, and 100 microM), tacrine (0.1, 1, and 10 microM), and rivastigmine (0.1, 1, and 10 microM) did not significantly decrease LDH release. The neuroprotective effect of donepezil was not antagonized by scopolamine or mecamylamine. These results demonstrate that donepezil has a protective effect against oxygen-glucose deprivation-induced injury to rat primary cultured cerebral cortical neurons. Besides, it is suggested that this effect of donepezil is independent of muscarinic cholinergic system and nicotinic cholinergic system. Thus, donepezil is expected to have a protective effect against progressive degeneration of brain neuronal cells in ischemic cerebrovascular disease and Alzheimer's disease.